Neuroprotective small organic molecules, compositions and uses of related thereto

ABSTRACT

The present application is directed to therapeutic compounds, compositions, and methods for culturing neuronal cells and for preventing and the treatment of neurodegenerative diseases, such as Parkinson&#39;s disease and amyotrophic lateral sclerosis (ALS).

RELATED APPLICATION

This application is a continuation application of, and claims thebenefit of, U.S. patent application Ser. No. 10/894,336, filed Jul. 19,2004 entitled “Neuroprotective Small Organic Molecules, Compositions andUses Related Thereto”, the contents of which are incorporated byreference herein in their entirety. No new matter has been added.

BACKGROUND OF THE INVENTION

The individual symptoms of Parkinson's disease have been described byphysicians from the time of Galen, but their occurrence as a syndromewas not recognized until 1817. In that year James Parkinson, a Londonphysician, published an essay in which he argued that several differentmotor symptoms could be considered together as a group forming adistinctive condition. His observations are interesting not only becausehis conclusion was correct but also because he made his observations inpart at a distance by watching the movements of Parkinsonian victims inthe street of London. Parkinson's disease has been called at differenttimes the shaking palsy or its Latin equivalent, paralysis agitans, butreceived its more common designation from Jean Charcot, who suggestedthat the disease be renamed to honor James Parkinson's recognition ofits essential nature.

Parkinson's disease is fairly common, estimates of its incidence varyingfrom 0.1 to 1.0% of the population. It is also of considerable interestfor a number of other reasons. First, the disease seems related to thedegeneration of the substantia nigra, and to the loss of theneurotransmitter substance dopamine, which is produced by cells of thisregion. The disease, therefore, provides an important insight into therole of this brainstem nucleus and its neurotransmitter in the controlof movement. Second, because a variety of pharmacological treatments forParkinson's disease relieve different features of its symptoms to someextent, the disease provides a model for understanding pharmacologicaltreatments of motor disorders in their more general aspects. Third,although Parkinson's disease is described as a disease entity, thesymptoms vary enormously among people, thus making manifest thecomplexity with which the components of movement are organized toproduce fluid motion. Fourth, because many of the symptoms ofParkinson's disease strikingly resemble changes in motor activity thatoccur as a consequence of aging, the disease provides indirect insightinto the more general problems of neural changes in aging.

There are three major types of Parkinson's disease: idiopathic,postencephalitic, and drug-induced. Parkinson's diseases may also resultfrom arteriosclerosis, may follow poisoning by carbon monoxide ormanganese intoxication, or may result from syphilis or the developmentof tumors. As is suggested by its name, the cause of idiopathicParkinson's disease is not known. Its origin may be familiar, or it maybe part of the aging process, but it is also widely thought that itmight have a viral origin. It most often occurs in people who are over50 years of age. The postencephalitic form originated in the sleepingsickness that appeared in the winter of 1916-1917 and vanished by 1927.Although the array of symptoms was bewilderingly varied, such thathardly any two patients seemed alike, Constantin von Economodemonstrated a unique pattern of brain damage associated with a virusinfection in the brains of patients who had died from the disease. Athird of those affected died in the acute stages of sleeping sickness instates either of coma or of sleeplessness. Although many people seemedto completely recover from the sickness, most subsequently developedneurological or psychiatric disorders and parkinsonism. The latencybetween the initial and subsequent occurrences of the disease has neverbeen adequately explained. Searches for viral particles orvirus-specific products in Parkinson patients have revealed no evidenceof viral cause. The third major cause of Parkinson's disease is morerecent, and is associated with ingestion of various drugs, particularlymajor tranquilizers that include reserpine and several phenothiazine andbutyrophenone derivatives. The symptoms are usually reversible, but theyare difficult to distinguish from those of the genuine disorder.

Recently it has been found that external agents can cause symptoms quiterapidly. Langston and coworkers have reported that a contaminant ofsynthetic heroin, MPTP, when taken by drug users is converted into MPP⁺,which is extremely toxic to dopamine cells. A number of young drug userswere found to display a complete parkinsonian syndrome after usingcontaminated drugs. This finding has suggested that other substancesmight cause similar effects. Demographic studies of patient admission inthe cities of Vancouver and Helsinki show an increase in the incidenceof patients getting the disease at ages younger than 40. This has raisedthe suggestion that water and air might contain environmental toxinsthat work in a fashion similar to MPTP.

Although Parkinsonian patients can be separated into clinical groups onthe basis of cause of the disease, it is nevertheless likely that themechanisms producing the symptoms have a common origin. Either thesubstrantia nigra is damaged, as occurs in idiopathic andpostencephalitic cases, or the activity of its cells is blocked or cellsare killed, as occurs in drug-induced parkinsonism. The cells of thesubstantia nigra contain a dark pigment in Parkinson's disease that isdepigmented by degeneration of the melatonin-containing neurons of thearea. The cells of the substantia nigra are the point of origin offibers that go to the basal ganglial frontal cortex and to the spinalcord. The neurotransmitter at the synapses of these projections isdopamine. It has been demonstrated by bioassay of the brains of deceasedparkinsonian patients, and by analysis of the major metabolite ofdopamine, homovanallic acid, which is excreted in the urine, that theamount of brain dopamine is reduced by over 90% and is often reduced toundetectable amounts. Thus, the cause of Parkinson's disease has beenidentified with some certainty as a lack of dopamine or, in drug-inducedcases, with a lack of dopamine action.

Accordingly, pharmaceuticals and methods of treatment for treatment orprophylaxis of Parkinson's disease are needed.

SUMMARY OF THE INVENTION

One aspect of the present application relates to a method for promotingthe survival of dopaminergic neurons or motoneurons by contacting thecells, in vitro or in vivo, with a compound as described herein in anamount effective to increase the rate of survival of the neuronsrelative to the absence of administration of the compound.

One aspect of the present application relates to a method for promotingthe survival of neurons of the substantia nigra by contacting the cells,in vitro or in vivo, with a compound as described herein in an amounteffective to increase the rate of survival of the neurons relative tothe absence of administration of the compound.

In other embodiments, the subject method can be used for protectingdopaminergic neurons and/or motoneurons of a mammal fromneurodegeneration, for preventing or treating neurodegenerativedisorder, for treatment of Parkinson's disease, and/or for treatment ofmotoneuron diseases such as amyotrophic lateral sclerosis (ALS).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-31 depict reactions useful for synthesizing compounds accordingto the present invention.

FIG. 32 shows the effects of compound A in midbrain cultures. Alltreatments were initiated at the time of plating. (A) Compound Astimulates dopamine (DA) uptake in a dose-dependent manner. DA uptakewas measured at 7 days in vitro (DIV) in the absence () or presence (o)of 10 μM GBR-12909. (B) Compound A promotes survival of TH⁺ neurons.Cultures were immunostained for TH at DIV 7 () or DIV 12 (o). *p<0.05,compared to corresponding untreated control, Student's t test. All datarepresent the average with standard deviation (SD) shown in error bars.In some instances, SDs are within the area of the symbols.

FIG. 33 illustrates the Selectivity of compound A in cultures ofmidbrain (A-E) and hindbrain (F, G). All treatments were initiated atthe time of plating. Where indicated, data represent percent ofuntreated control (Con). (A) Compound A does not increase total numberof NSE⁺ cells. Cultures were left untreated (solid bars) or treated with2.5 μM compound A (open bars), fixed at DIV 0 (1 hr), 5 or 7, andstained with anti-NSE. (B) Unlike forskolin (Fk), compound A does notaffect GFAP⁺ cell number. Cultures were treated with compound A or 25 μMFk, and stained with anti-GFAP at DIV 7. (C, D) Addition of compound Adoes not lead to an increase in GABA or 5-HT uptake. Cells were treatedwith fetal bovine serum (FBS, 2% as positive control) or compound A.Uptake was measured at DIV 8. (F) Compound A treatment does produce anincrease in islet-1⁺ cell numbers. Cultures were left untreated (solidbars) or treated with 2.5 μM compound A (open bars), stained withislet-1 at DIV 1, 2, and 3. *p<0.05 versus corresponding untreatedcultures (solid bar); p<0.05 between DIV, two-way ANOVA test. (F, G)Compound A treatment does not change either the number of TPH⁺ cells northe amount of serotonin uptake in hindbrain cultures. Cells were treatedwith FBS (2% as positive control) or compound A, stained with anti-TPHat DIV 13 and counted (F). 5-HT uptake was measured at DIV 8 (G). Alldata represent averages (n=2) with SD shown in error bars.

FIG. 34 depicts mechanism of compound A effect in midbrain cultures. 2.5μM compound A was added at the time of plating. (A) Compound A reducesthe rate of apoptosis of TH-positive cells. *p<0.05 versus Con,Student's t test. (B) Compound A protects TH-positive cells from MPP⁺induced neurotoxicity. Cultures at DIV 4 were treated with 2 μM MPP⁺(open bars) for 36 hrs, and processed for TH staining. *p<0.05 versuscorresponding cultures not treated with MPP⁺ (solid bars). #p<0.05versus corresponding cultures without Compound A, Student's t test. Datarepresent averages (n=3) with SD shown in error bars.

FIG. 35 depicts the additive effect of conjoint administration ofcompound A with GDNF. Cells were treated with GDNF in the absence (o) orpresence () of 2.5 μM compound A. Each data point is the average ofduplicate assays. Data represent percent of untreated control. *p<0.05compared to corresponding GDNF alone treatment, Student's t test.

FIG. 36 illustrates the effects of compounds X′, Y′ and Z′ on dopamineuptake as compared to compound A. Each data point is the average ofduplicate assays. SDs are within 15% and are shown in error bars incompound X′ only. The 3 analogs all show significant differences (p<0.05versus untreated control) at concentrations 0.16 μM and up. Compound Z′also shows p<0.05 at 0.04 μM.

DETAILED DESCRIPTION OF THE INVENTION

The subject compounds, as described in detail below, are useful asprotective agents in the treatment and prophylaxis of neurodegenerativedisorders, particularly those resulting from the loss of dopaminergicneurons and/or motoneurons, or the general loss tissue from thesubstantia nigra. As described with greater detail below, exemplarydisorders (“candidate disorders”) suitable for treatment with subjectinhibitors include Parkinson's disease, amyotrophic lateral sclerosis(ALS) and the like. In terms of treatment, once a patient experiencessymptoms of a candidate disorder, a goal of therapy is prevention offurther loss of neuron function.

The subject invention also utilizes such compounds as cell cultureadditives for the maintenance of differentiated neurons in cultures,e.g., in cultures of dopaminergic neurons and motoneurons. The subjectmethods and compositions can also be used to augment the implantation ofsuch neuronal cells in an animal.

I. Overview

The present application is directed to compositions and methods for thetreatment and prophylaxis of neurodegenerative disorders, particularlythose resulting from the loss of dopaminergic neurons and/ormotoneurons, or the general loss tissue from the substantia nigra. Thesubject methods are effective on both human and animal subjectsafflicted with these conditions. Animal subjects include both domesticanimals and livestock, raised either as pets or for commercial purposes,such as dogs, cats, cattle, horses, sheep, hogs, and goats.

II. Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The term “ED₅₀” means the dose of a drug that produces 50% of itsmaximum response or effect.

An “effective amount” of a subject compound, with respect to the subjectmethod of treatment, refers to an amount of the therapeutic in apreparation which, when applied as part of a desired dosage regimencauses a increase in survival of a neuronal cell population according toclinically acceptable standards for the treatment or prophylaxis of aparticular disorder.

The term “LD₅₀” means the dose of a drug that is lethal in 50% of testsubjects.

A “patient” or “subject” to be treated by the subject method aremammals, including humans.

By “prevent degeneration” it is meant reduction in the loss of cells(such as from apoptosis), or reduction in impairment of cell function,e.g., release of dopamine in the case of dopaminergic neurons.Generally, as used herein, a therapeutic that “prevents” a disorder orcondition refers to a compound that, in a sample, reduces the occurrenceof the disorder or condition in the sample, relative to an untreatedcontrol sample, or delays the onset of one or more symptoms of thedisorder or condition.

The term “prodrug” is intended to encompass compounds that, underphysiological conditions, are converted into the therapeutically activeagents of the present invention. A common method for making a prodrug isto include selected moieties that are hydrolyzed under physiologicalconditions to reveal the desired molecule. In other embodiments, theprodrug is converted by an enzymatic activity of the host animal.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

A “trophic factor” is a molecule that directly or indirectly affects thesurvival or function of a neuronal cell, e.g., a dopaminergic cell ormotoneuron.

A “trophic amount” of a subject compound is an amount sufficient to,under the circumstances, cause an increase in the rate of survival orthe functional performance of a neuronal cell, e.g., a dopaminergicneuron or motoneuron.

‘Acyl’ refers to a group suitable for acylating a nitrogen atom to forman amide or carbamate, a carbon atom to form a ketone, a sulfur atom toform a thioester, or an oxygen atom to form an ester group, e.g., ahydrocarbon attached to a —C(═O)— moiety. Preferred acyl groups includebenzoyl, acetyl, tert-butyl acetyl, pivaloyl, and trifluoroacetyl. Morepreferred acyl groups include acetyl and benzoyl. The most preferredacyl group is acetyl.

The term ‘acylamino’ is art-recognized and preferably refers to a moietythat can be represented by the general formula:

wherein R₉ and R′₁₁ each independently represent hydrogen or ahydrocarbon substituent, such as alkyl, heteroalkyl, aryl, heteroaryl,carbocyclic aliphatic, and heterocyclic aliphatic.

The terms ‘amine’ and ‘amino’ are art-recognized and refer to bothunsubstituted and substituted amines as well as ammonium salts, e.g., ascan be represented by the general formula:

wherein R₉, R₁₀, and R′₁₀ each independently represent hydrogen or ahydrocarbon substituent, or R₉ and R₁₀ taken together with the N atom towhich they are attached complete a heterocycle having from 4 to 8 atomsin the ring structure. In preferred embodiments, none of R₉, R₁₀, andR′₁₀ is acyl, e.g., R₉, R₁₀, and R′₁₀ are selected from hydrogen, alkyl,heteroalkyl, aryl, heteroaryl, carbocyclic aliphatic, and heterocyclicaliphatic. The term ‘alkylamine’ as used herein means an amine group, asdefined above, having at least one substituted or unsubstituted alkylattached thereto. Amino groups that are positively charged (e.g., R′₁₀is present) are referred to as ‘ammonium’ groups. In amino groups otherthan ammonium groups, the amine is preferably basic, e.g., its conjugateacid has a pK_(a) above 7.

The terms ‘amido’ and ‘amide’ are art-recognized as an amino-substitutedcarbonyl, such as a moiety that can be represented by the generalformula:

wherein R₉ and R₁₀ are as defined above. In certain embodiments, theamide will include imides.

‘Alkyl’ refers to a saturated or unsaturated hydrocarbon chain having 1to 18 carbon atoms, preferably 1 to 12, more preferably 1 to 6, morepreferably still 1 to 4 carbon atoms. Alkyl chains may be straight(e.g., n-butyl) or branched (e.g., sec-butyl, isobutyl, or t-butyl).Preferred branched alkyls have one or two branches, preferably onebranch. Preferred alkyls are saturated. Unsaturated alkyls have one ormore double bonds and/or one or more triple bonds. Preferred unsaturatedalkyls have one or two double bonds or one triple bond, more preferablyone double bond. Alkyl chains may be unsubstituted or substituted withfrom 1 to 4 substituents. Preferred alkyls are unsubstituted. Preferredsubstituted alkyls are mono-, di-, or trisubstituted. Preferred alkylsubstituents include halo, haloalkyl, hydroxy, aryl (e.g., phenyl,tolyl, alkoxyphenyl, alkyloxycarbonylphenyl, halophenyl), heterocyclyl,and heteroaryl.

The terms ‘alkenyl’ and ‘alkynyl’ refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond,respectively. When not otherwise indicated, the terms alkenyl andalkynyl preferably refer to lower alkenyl and lower alkynyl groups,respectively. When the term alkyl is present in a list with the termsalkenyl and alkynyl, the term alkyl refers to saturated alkyls exclusiveof alkenyls and alkynyls.

The terms ‘alkoxyl’ and ‘alkoxy’ as used herein refer to an —O-alkylgroup. Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy, and the like. An ‘ether’ is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of a hydrocarbon thatrenders that hydrocarbon an ether can be an alkoxyl, or another moietysuch as —O-aryl, —O-heteroaryl, —O-heteroalkyl, —O-aralkyl,—O-heteroaralkyl, —O-carbocylic aliphatic, or —O-heterocyclic aliphatic.

An ‘alkylseleno’ or ‘selenoalkyl’ refers to a —Se-alkyl group.‘Selenoethers’ more broadly refers to two hydrocarbon groups linked by aselenium atom. Accordingly, the substituent of a hydrocarbon thatrenders that hydrocarbon a selenoether can be an alkylseleno, or anothermoiety such as —Se-aryl, —Se-heteroaryl, —Se-heteroalkyl, —Se-aralkyl,—Se-heteroaralkyl, —Se-carbocylic aliphatic, or —Se-heterocyclicaliphatic.

The term ‘alkylthio’ refers to an —S-alkyl group. Representativealkylthio groups include methylthio, ethylthio, and the like.‘Thioether’ refers to a sulfur atom bound to two hydrocarbonsubstituents, e.g., an ether wherein the oxygen is replaced by sulfur.Thus, a thioether substituent on a carbon atom refers to ahydrocarbon-substituted sulfur atom substituent, such as alkylthio orarylthio, etc.

The term ‘aralkyl’, as used herein, refers to an alkyl group substitutedwith an aryl group.

‘Aryl ring’ refers to an aromatic hydrocarbon ring system. Aromaticrings are monocyclic or fused bicyclic ring systems, such as phenyl,naphthyl, etc. Monocyclic aromatic rings contain from about 5 to about10 carbon atoms, preferably from 5 to 7 carbon atoms, and mostpreferably from 5 to 6 carbon atoms in the ring. Bicyclic aromatic ringscontain from 8 to 12 carbon atoms, preferably 9 or 10 carbon atoms inthe ring. The term ‘aryl’ also includes bicyclic ring systems whereinonly one of the rings is aromatic, e.g., the other ring is cycloalkyl,cycloalkenyl, or heterocyclyl. Aromatic rings may be unsubstituted orsubstituted with from 1 to about 5 substituents on the ring. Preferredaromatic ring substituents include: halo, cyano, lower alkyl,heteroalkyl, haloalkyl, phenyl, phenoxy, or any combination thereof.More preferred substituents include lower alkyl, cyano, halo, andhaloalkyl.

‘Biohydrolyzable amide’ refers to an amide moiety that is cleaved (e.g.,to form a hydroxyl and a carboxylic acid) under physiologicalconditions. Physiological conditions include the acidic and basicenvironments of the digestive tract (e.g., stomach, intestines, etc.),enzymatic cleavage, metabolism, and other biological processes, andpreferably refer to physiological conditions in a vertebrate, such as amammal.

‘Biohydrolyzable ester’ refers to an ester moiety that is cleaved (e.g.,to form a hydroxyl and a carboxylic acid) under physiologicalconditions. Physiological conditions include the acidic and basicenvironments of the digestive tract (e.g., stomach, intestines, etc.),enzymatic cleavage, metabolism, and other biological processes, andpreferably refer to physiological conditions in a vertebrate, such as amammal.

‘Biohydrolyzable imide’ refers to an imide moiety that is cleaved (e.g.,to form a hydroxyl and a carboxylic acid) under physiologicalconditions. Physiological conditions include the acidic and basicenvironments of the digestive tract (e.g., stomach, intestines, etc.),enzymatic cleavage, metabolism, and other biological processes, andpreferably refer to physiological conditions in a vertebrate, such as amammal.

‘Carbocyclic aliphatic ring’ refers to a saturated or unsaturatedhydrocarbon ring. Carbocyclic aliphatic rings are not aromatic.Carbocyclic aliphatic rings are monocyclic, or are fused, spiro, orbridged bicyclic ring systems. Monocyclic carbocyclic aliphatic ringscontain from about 4 to about 10 carbon atoms, preferably from 4 to 7carbon atoms, and most preferably from 5 to 6 carbon atoms in the ring.Bicyclic carbocyclic aliphatic rings contain from 8 to 12 carbon atoms,preferably from 9 to 10 carbon atoms in the ring. Carbocyclic aliphaticrings may be unsubstituted or substituted with from 1 to 4 substituentson the ring. Preferred carbocyclic aliphatic ring substituents includehalo, cyano, alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or anycombination thereof. More preferred substituents include halo andhaloalkyl. Preferred carbocyclic aliphatic rings include cyclopentyl,cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. More preferredcarbocyclic aliphatic rings include cyclohexyl, cycloheptyl, andcyclooctyl.

The term ‘carbonyl’ is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, hydrocarbon substituent, or a pharmaceuticallyacceptable salt, R_(11′) represents a hydrogen or hydrocarbonsubstituent. Where X is an oxygen and R₁₁ or R_(11′) is not hydrogen,the formula represents an ‘ester’. Where X is an oxygen, and R₁₁ is asdefined above, the moiety is referred to herein as a carboxyl group, andparticularly when R_(11′) is a hydrogen, the formula represents a‘carboxylic acid’. Where X is an oxygen, and R_(11′) is hydrogen, theformula represents a ‘formate’. In general, where the oxygen atom of theabove Formula IIs replaced by sulfur, the formula represents a‘thiocarbonyl’ group. Where X is a sulfur and R₁₁ or R_(11′) is nothydrogen, the formula represents a ‘thioester.’ Where X is a sulfur andR₁₁ is hydrogen, the formula represents a ‘thiocarboxylic acid.’ Where Xis a sulfur and R_(11′) is hydrogen, the formula represents a‘thioformate.’ On the other hand, where X is a bond, R₁₁ is nothydrogen, and the carbonyl is bound to a hydrocarbon, the above formularepresents a ‘ketone’ group. Where X is a bond, R₁₁ is hydrogen, and thecarbonyl is bound to a hydrocarbon, the above formula represents an‘aldehyde’ or ‘formyl’ group.

‘Ci alkyl’ is a heteroalkyl chain having i member atoms. For example, C4alkyls contain four carbon member atoms. C4 alkyls containing may besaturated or unsaturated with one or two double bonds (cis or trans) orone triple bond. Preferred C4 alkyls are saturated. Preferredunsaturated C4 alkyl have one double bond. C4 alkyl may be unsubstitutedor substituted with one or two substituents. Preferred substituentsinclude lower alkyl, lower heteroalkyl, cyano, halo, and haloalkyl.

‘Halogen’ refers to fluoro, chloro, bromo, or iodo substituents.Preferred halo are fluoro, chloro and bromo; more preferred are chloroand fluoro.

‘Haloalkyl’ refers to a straight, branched, or cyclic hydrocarbonsubstituted with one or more halo substituents. Preferred haloalkyl areC1-C12; more preferred are C1-C6; more preferred still are C1-C3.Preferred halo substituents are fluoro and chloro. The most preferredhaloalkyl is trifluoromethyl.

‘Heteroalkyl’ is a saturated or unsaturated chain of carbon atoms and atleast one heteroatom, wherein no two heteroatoms are adjacent.Heteroalkyl chains contain from 1 to 18 member atoms (carbon andheteroatoms) in the chain, preferably 1 to 12, more preferably 1 to 6,more preferably still 1 to 4. Heteroalkyl chains may be straight orbranched. Preferred branched heteroalkyl have one or two branches,preferably one branch. Preferred heteroalkyl are saturated. Unsaturatedheteroalkyl have one or more double bonds and/or one or more triplebonds. Preferred unsaturated heteroalkyl have one or two double bonds orone triple bond, more preferably one double bond. Heteroalkyl chains maybe unsubstituted or substituted with from 1 to about 4 substituentsunless otherwise specified. Preferred heteroalkyl are unsubstituted.Preferred heteroalkyl substituents include halo, aryl (e.g., phenyl,tolyl, alkoxyphenyl, alkoxycarbonylphenyl, halophenyl), heterocyclyl,heteroaryl. For example, alkyl chains substituted with the followingsubstituents are heteroalkyl: alkoxy (e.g., methoxy, ethoxy, propoxy,butoxy, pentoxy), aryloxy (e.g., phenoxy, chlorophenoxy, tolyloxy,methoxyphenoxy, benzyloxy, alkoxycarbonylphenoxy, acyloxyphenoxy),acyloxy (e.g., propionyloxy, benzoyloxy, acetoxy), carbamoyloxy,carboxy, mercapto, alkylthio, acylthio, arylthio (e.g., phenylthio,chlorophenylthio, alkylphenylthio, alkoxyphenylthio, benzylthio,alkoxycarbonylphenylthio), amino (e.g., amino, mono- and di-C1-C3alkylamino, methylphenylamino, methylbenzylamino, C1-C3 alkylamido,carbamamido, ureido, guanidino).

‘Heteroatom’ refers to a multivalent non-carbon atom, such as a boron,phosphorous, silicon, nitrogen, sulfur, or oxygen atom, preferably anitrogen, sulfur, or oxygen atom. Groups containing more than oneheteroatom may contain different heteroatoms.

‘Heteroaryl ring’ refers to an aromatic ring system containing carbonand from 1 to about 4 heteroatoms in the ring. Heteroaromatic rings aremonocyclic or fused bicyclic ring systems. Monocyclic heteroaromaticrings contain from about 5 to about 10 member atoms (carbon andheteroatoms), preferably from 5 to 7, and most preferably from 5 to 6 inthe ring. Bicyclic heteroaromatic rings contain from 8 to 12 memberatoms, preferably 9 or 10 member atoms in the ring. The term‘heteroaryl’ also includes bicyclic ring systems wherein only one of therings is aromatic, e.g., the other ring is cycloalkyl, cycloalkenyl, orheterocyclyl. Heteroaromatic rings may be unsubstituted or substitutedwith from 1 to about 4 substituents on the ring. Preferredheteroaromatic ring substituents include halo, cyano, lower alkyl,heteroalkyl, haloalkyl, phenyl, phenoxy or any combination thereof.Preferred heteroaromatic rings include thienyl, thiazolyl, oxazolyl,pyrrolyl, purinyl, pyrimidyl, pyridyl, and furanyl. More preferredheteroaromatic rings include thienyl, furanyl, and pyridyl.

‘Heterocyclic aliphatic ring’ is a non-aromatic saturated or unsaturatedring containing carbon and from 1 to about 4 heteroatoms in the ring,wherein no two heteroatoms are adjacent in the ring and preferably nocarbon in the ring attached to a heteroatom also has a hydroxyl, amino,or thiol group attached to it. Heterocyclic aliphatic rings aremonocyclic, or are fused or bridged bicyclic ring systems. Monocyclicheterocyclic aliphatic rings contain from about 4 to about 10 memberatoms (carbon and heteroatoms), preferably from 4 to 7, and mostpreferably from 5 to 6 member atoms in the ring. Bicyclic heterocyclicaliphatic rings contain from 8 to 12 member atoms, preferably 9 or 10member atoms in the ring. Heterocyclic aliphatic rings may beunsubstituted or substituted with from 1 to about 4 substituents on thering. Preferred heterocyclic aliphatic ring substituents include halo,cyano, lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or anycombination thereof. More preferred substituents include halo andhaloalkyl. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, hydantoin,oxazoline, imidazolinetrione, triazolinone, quinoline, phthalazine,naphthyridine, quinoxaline, quinazoline, quinoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,phenazine, phenarsazine, phenothiazine, furazan, phenoxazine,pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine,morpholine, lactones, lactams such as azetidinones and pyrrolidinones,sultams, sultones, and the like. Preferred heterocyclic aliphatic ringsinclude piperazyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl andpiperidyl. Heterocycles can also be polycycles.

The term ‘hydroxyl’ means —OH.

‘Lower alkyl’ refers to an alkyl chain comprised of 1 to 5, preferably 1to 4 carbon member atoms, more preferably 1 or 2 carbon member atoms.Lower alkyls may be saturated or unsaturated. Preferred lower alkyls aresaturated. Lower alkyls may be unsubstituted or substituted with one orabout two substituents. Preferred substituents on lower alkyl includecyano, halo, trifluoromethyl, amino, and hydroxyl. Throughout theapplication, preferred alkyl groups are lower alkyls. In preferredembodiments, a substituent designated herein as alkyl is a lower alkyl.Likewise, ‘lower alkenyl’ and ‘lower alkynyl’ have similar chainlengths.

‘Lower heteroalkyl’ refers to a heteroalkyl chain comprised of 1 to 4,preferably 1 to 3 member atoms, more preferably 1 to 2 member atoms.Lower heteroalkyl contain one or two non-adjacent heteroatom memberatoms. Preferred lower heteroalkyl contain one heteroatom member atom.Lower heteroalkyl may be saturated or unsaturated. Preferred lowerheteroalkyl are saturated. Lower heteroalkyl may be unsubstituted orsubstituted with one or about two substituents. Preferred substituentson lower heteroalkyl include cyano, halo, trifluoromethyl, and hydroxyl.

‘Mi heteroalkyl’ is a heteroalkyl chain having i member atoms. Forexample, M4 heteroalkyls contain one or two non-adjacent heteroatommember atoms. M4 heteroalkyls containing 1 heteroatom member atom may besaturated or unsaturated with one double bond (cis or trans) or onetriple bond. Preferred M4 heteroalkyl containing 2 heteroatom memberatoms are saturated. Preferred unsaturated M4 heteroalkyl have onedouble bond. M4 heteroalkyl may be unsubstituted or substituted with oneor two substituents. Preferred substituents include lower alkyl, lowerheteroalkyl, cyano, halo, and haloalkyl.

‘Member atom’ refers to a polyvalent atom (e.g., C, O, N, or S atom) ina chain or ring system that constitutes a part of the chain or ring. Forexample, in cresol, six carbon atoms are member atoms of the ring andthe oxygen atom and the carbon atom of the methyl substituent are notmember atoms of the ring.

As used herein, the term ‘nitro’ means —NO₂.

‘Pharmaceutically acceptable salt’ refers to a cationic salt formed atany acidic (e.g., hydroxamic or carboxylic acid) group, or an anionicsalt formed at any basic (e.g., amino or guanidino) group. Such saltsare well known in the art. See e.g., World Patent Publication 87/05297,Johnston et al., published Sep. 11, 1987, incorporated herein byreference. Such salts are made by methods known to one of ordinary skillin the art. It is recognized that the skilled artisan may prefer onesalt over another for improved solubility, stability, formulation ease,price and the like. Determination and optimization of such salts iswithin the purview of the skilled artisan's practice. Preferred cationsinclude the alkali metals (such as sodium and potassium), and alkalineearth metals (such as magnesium and calcium) and organic cations, suchas trimethylammonium, tetrabutylammonium, etc. Preferred anions includehalides (such as chloride), sulfonates, carboxylates, phosphates, andthe like. Clearly contemplated in such salts are addition salts that mayprovide an optical center where once there was none. For example, achiral tartrate salt may be prepared from the compounds of theinvention. This definition includes such chiral salts.

‘Phenyl’ is a six-membered monocyclic aromatic ring that may or may notbe substituted with from 1 to 5 substituents. The substituents may belocated at the ortho, meta or para position on the phenyl ring, or anycombination thereof. Preferred phenyl substituents include: halo, cyano,lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or any combinationthereof. More preferred substituents on the phenyl ring include halo andhaloalkyl. The most preferred substituent is halo.

The terms ‘polycyclyl’ and ‘polycyclic group’ refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, heteroaryls, aryls and/orheterocyclyls) in which two or more member atoms of one ring are memberatoms of a second ring. Rings that are joined through non-adjacent atomsare termed ‘bridged’ rings, and rings that are joined through adjacentatoms are ‘fused rings’.

The term ‘sulfhydryl’ means —SH, and the term ‘sulfonyl’ means —SO₂—.

The term ‘sulfamoyl’ is art-recognized and includes a moiety representedby the general formula:

in which R₉ and R₁₀ are as defined above.

The term ‘sulfate’ is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₁₀ is as defined above.

The term ‘sulfonamido’ is art-recognized, and includes a moietyrepresented by the general formula:

in which R₉ and R′₁₁ are as defined above.

The terms ‘sulfoxido’ and ‘sulfinyl’, as used herein, are art-recognizedand include a moiety represented by the general formula:

in which R₉ is as defined above.

A ‘substitution’ or ‘substituent’ on a small organic molecule generallyrefers to a position on a multi-valent atom bound to a moiety other thanhydrogen, e.g., a position on a chain or ring exclusive of the memberatoms of the chain or ring. Such moieties include those defined hereinand others as are known in the art, for example, halogen, alkyl,alkenyl, alkynyl, azide, haloalkyl, hydroxyl, carbonyl (such ascarboxyl, alkoxycarbonyl, formyl, ketone, or acyl), thiocarbonyl (suchas thioester, thioacetate, or thioformate), alkoxyl, phosphoryl,phosphonate, phosphinate, amine, amide, amidine, imine, cyano, nitro,azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl,sulfonamido, sulfonyl, silyl, ether, cycloalkyl, heterocyclyl,heteroalkyl, heteroalkenyl, and heteroalkynyl, heteroaralkyl, aralkyl,aryl or heteroaryl. It will be understood by those skilled in the artthat certain substituents, such as aryl, heteroaryl, polycyclyl, alkoxy,alkylamino, alkyl, cycloalkyl, heterocyclyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, and heteroalkynyl, can themselves besubstituted, if appropriate. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds. It will be understood that ‘substitution’ or ‘substitutedwith’ includes the implicit proviso that such substitution is inaccordance with permitted valence of the substituted atom and thesubstituent, and that the substitution results in a stable compound,e.g., which does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, hydrolysis, etc.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl, and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

The phrase ‘protecting group’ as used herein means temporarysubstituents that protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991; and Kocienski, P. J. Protecting Groups, Georg Thieme Verlag: NewYork, 1994).

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term ‘hydrocarbon’ is contemplatedto include all permissible compounds or moieties having at least onecarbon-hydrogen bond. In a broad aspect, the permissible hydrocarbonsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivatization with a chiral auxiliary, where the resultingdiastereomeric mixture is separated and the auxiliary group cleaved toprovide the pure desired enantiomers. Alternatively, where the moleculecontains a basic functional group, such as amino, or an acidicfunctional group, such as carboxyl, diastereomeric salts can be formedwith an appropriate optically active acid or base, followed byresolution of the diastereomers thus formed by fractionalcrystallization or chromatographic means well known in the art, andsubsequent recovery of the purified enantiomers. Enantiomers may also beseparated using a ‘chiral column’, i.e., by chromatographicallyseparating the enantiomers using chiral molecules bound to a solidsupport.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the sameuseful properties thereof, wherein one or more simple variations ofsubstituents are made which do not adversely affect the efficacy of thecompound. In general, the compounds of the present invention may beprepared by the methods illustrated in the general reaction schemes as,for example, described below, or by modifications thereof, using readilyavailable starting materials, reagents and conventional synthesisprocedures. In these reactions, it is also possible to make use ofvariants that are in themselves known, but are not mentioned here.

III. Exemplary Applications of Method and Compositions

One aspect of the present invention relates to a method of maintaining adifferentiated state, e.g., enhancing survival, of a neuronal cell, bycontacting the cells with a trophic amount of a subject compound. Forinstance, it is contemplated by the invention that, the subject methodcould be used to maintain differentiated neuronal tissue both in vitroand in vivo.

The present method is applicable to cell culture techniques. In vitroneuronal culture systems have proved to be fundamental and indispensabletools for the study of neural development, as well as the identificationof neurotrophic factors such as nerve growth factor (NGF), ciliaryneurotrophic factors (CNTF) (see Lin et al. Science 1989, 246,1023-1025), glial cell line-derived neurotrophic factor (GDNF) (see Linet al. Science 1993, 260, 1130-1132), and brain-derived neurotrophicfactor (BDNF) (see Leibrock et al. Nature 1989, 341, 149-152). Once aneuronal cell has become terminally differentiated it typically will notchange to another terminally differentiated cell-type. However, neuronalcells can nevertheless readily lose their differentiated state. This iscommonly observed when they are grown in culture from adult tissue, andwhen they form a blastema during regeneration. The present methodprovides a means for ensuring an adequately restrictive environment inorder to maintain dopaminergic neuron and motoneuron in differentiatedstates, and can be employed, for instance, in cell cultures designed totest the specific activities of other trophic factors.

In such embodiments of the subject method, a culture of differentiatedcells including dopaminergic neurons and/or motoneurons can be contactedwith a subject compound in order to maintain the integrity of a cultureof terminally differentiated neuronal cells by preventing loss ofdifferentiation. Such neuronal cultures can be used as convenient assaysystems as well as sources of implantable cells for therapeutictreatments.

The subject method can be used in conjunction with agents that inducethe differentiation of neuronal precursors, e.g., progenitor or stemcells, into dopaminergic neurons or motoneurons.

Cells can be obtained from embryonic, post-natal, juvenile or adultneural tissue from any animal. By any animal is meant any multicellularanimal that contains nervous tissue. More particularly, is meant anyfish, reptile, bird, amphibian or mammal and the like. The mostpreferable donors are mammals, especially humans and non-human primates,pigs, cows, and rodents.

Intracerebral neural grafting has emerged recently as an additionalpotential to CNS therapy. For example, one approach to repairing damagedbrain tissues involves the transplantation of cells from fetal orneonatal animals into the adult brain (Dunnett et al. J Exp Biol 1987,123, 265-289; and Freund et al. J Neurosci 1985. 5, 603-616). Fetalneurons from a variety of brain regions can be successfully incorporatedinto the adult brain, and such grafts can alleviate behavioral defects.For example, movement disorder induced by lesions of dopaminergicprojections to the basal ganglia can be prevented by grafts of embryonicdopaminergic neurons. Complex cognitive functions that are impairedafter lesions of the neocortex can also be partially restored by graftsof embryonic cortical cells. Transplantation of fetal brain cells, whichcontain precursors of the dopaminergic neurons, has been examined withsuccess as a treatment for Parkinson's disease. In animal models and inpatients with this disease, fetal brain cell transplantations haveresulted in the reduction of motor abnormalities. Furthermore, itappears that the implanted fetal dopaminergic neurons form synapses withsurrounding host neurons. However, in the art, the transplantation offetal brain cells is limited due, for example, to the limited survivaltime of the implanted neuronal precursors and differentiated neuronsarising therefrom. The subject invention provides a means for extendingthe usefulness of such transplants by enhancing the survival ofdopaminergic neurons and/or motoneurons in the transplant.

In the specific case of Parkinson's disease, treatment with a subjectcompound can improve the in vivo survival of fetal and adultdopaminergic neurons, and thus can provide a more effective treatment ofthis disease.

In the case of a heterologous donor animal, the animal may beeuthanized, and the brain and specific area of interest removed using asterile procedure. Brain areas of particular interest include any areafrom which progenitor cells can be obtained which will providedopaminergic neurons or motoneurons upon differentiation. These regionsinclude areas of the central nervous system (CNS) including thesubstantia nigra pars compacta, which is found to be degenerated inParkinson's Disease patients.

Human heterologous neural progenitor cells may be derived from fetaltissue obtained from elective abortion, or from a post-natal, juvenileor adult organ donor. Autologous neural tissue can be obtained bybiopsy, or from patients undergoing neurosurgery in which neural tissueis removed, such as during epilepsy surgery.

Cells can be obtained from donor tissue by dissociation of individualcells from the connecting extracellular matrix of the tissue.Dissociation can be obtained using any known procedure, includingtreatment with enzymes such as trypsin, collagenase and the like, or byusing physical methods of dissociation such as with a blunt instrument.Dissociation of fetal cells can be carried out in tissue culture medium,while a preferable medium for dissociation of juvenile and adult cellsis artificial cerebral spinal fluid (aCSF). Regular aCSF contains 124 mMNaCl, 5 mM KCl, 1.3 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃, and 10 mMD-glucose. Low Ca²⁺ aCSF contains the same ingredients except for MgCl₂at a concentration of 3.2 mM and CaCl₂ at a concentration of 0.1 mM.

Dissociated cells can be placed into any known culture medium capable ofsupporting cell growth, including MEM, DMEM, RPMI, F-12, and the like,containing supplements which are required for cellular metabolism suchas glutamine and other amino acids, vitamins, minerals and usefulproteins such as transferrin and the like. Medium may also containantibiotics to prevent contamination with yeast, bacteria and fungi suchas penicillin, streptomycin, gentamicin and the like. In some cases, themedium may contain serum derived from bovine, equine, chicken and thelike. A particularly preferable medium for cells is a mixture of DMEMand F-12.

Conditions for culturing should be close to physiological conditions.The pH of the culture media should be close to physiological pH,preferably between pH 6-8, more preferably close to pH 7, even moreparticularly about pH 7.4. Cells should be cultured at a temperatureclose to physiological temperature, preferably between 30° C.-40° C.,more preferably between 32° C.-38° C., and most preferably between 35°C.-37° C.

Cells can be grown in suspension or on a fixed substrate, butproliferation of the progenitors is preferably done in suspension togenerate large numbers of cells by formation of “neurospheres” (see, forexample, Reynolds et al. Science 1992, 255, 1070-1709; and PCTPublications WO93/01275, WO94/09119, WO94/10292, and WO94/16718). In thecase of propagating (or splitting) suspension cells, flasks are shakenwell and the neurospheres allowed to settle on the bottom corner of theflask. The spheres are then transferred to a 50 ml centrifuge tube andcentrifuged at low speed. The medium is aspirated, the cells resuspendedin a small amount of medium with growth factor, and the cellsmechanically dissociated and resuspended in separate aliquots of media.

Cell suspensions in culture medium are supplemented with any growthfactor that allows for the proliferation of progenitor cells and seededin any receptacle capable of sustaining cells, though as set out above,preferably in culture flasks or roller bottles. Cells typicallyproliferate within 3-4 days in a 37° C. incubator, and proliferation canbe reinitiated at any time after that by dissociation of the cells andresuspension in fresh medium containing growth factors.

In the absence of substrate, cells lift off the floor of the flask andcontinue to proliferate in suspension forming a hollow sphere ofundifferentiated cells. After approximately 3-10 days in vitro, theproliferating clusters (neurospheres) are fed every 2-7 days, and moreparticularly every 2-4 days by gentle centrifugation and resuspension inmedium containing growth factor.

After 6-7 days in vitro, individual cells in the neurospheres can beseparated by physical dissociation of the neurospheres with a bluntinstrument, more particularly by triturating the neurospheres with apipette. Single cells from the dissociated neurospheres are suspended inculture medium containing growth factors, and differentiation of thecells can be induced by plating (or resuspending) the cells in thepresence of a factor capable of sustaining differentiation, e.g., suchas a subject compound of the present invention.

Stem cells useful in the present invention are generally known. Forexample, several neural crest cells have been identified, some of whichare multipotent and likely represent uncommitted neural crest cells. Therole of subject compounds employed in the present method to culture suchstem cells is to maintain differentiation a committed progenitor celland/or a terminally differentiated dopaminergic neuron or motoneuron.The subject compound can be used alone, or can be used in combinationwith other neurotrophic factors that act to more particularly enhance aparticular differentiation fate of the neuronal progenitor cell.

In addition to the implantation of cells cultured in the presence of asubject compound and other in vitro uses described above, yet anotheraspect of the present invention concerns the therapeutic application ofa subject compound to enhance survival of dopaminergic neurons andmotoneurons in vivo. The ability of a subject compound to maintainneuronal differentiation of dopaminergic neuron and motoneuron indicatesthat such compounds can reasonably be expected to facilitate control ofthese neuronal cell-types in adult tissue with regard to maintenance,functional performance, aging and prevention of degeneration andpremature death which result from loss of differentiation in certainpathological conditions. In light of this understanding, the presentinvention specifically contemplates applications of the subject methodto the treatment of (prevention and/or reduction of the severity of)neurological conditions deriving from (i) loss of dopaminergic cells,(ii) loss of motoneurons, and/or (iii) loss of neurons of the substantianigra. In this regard, the subject method is useful in the treatment ofchronic neurodegenerative diseases of the nervous system, includingParkinson's disease, amylotrophic lateral sclerosis and the like.

Many neurological disorders are associated with degeneration of discretepopulations of neuronal elements and may be treatable with a therapeuticregimen that includes a compound according to the subject invention. Asdescribed in the appended examples, subject compounds exert trophic andsurvival-promoting actions on substantia nigra dopaminergic neurons. Invivo, treatment with a subject compound is expected to stimulate thedopaminergic phenotype of substantia nigra neurons and restorefunctional deficits induced by axotomy or dopaminergic neurotoxins, andmay be used the treatment of Parkinson's disease, a neurodegenerativedisease characterized by the loss of dopaminergic neurons. Thus, in oneembodiment, the subject method comprises administering to an animalafflicted with Parkinson's disease, or at risk of developing Parkinson'sdisease, an amount of a subject compound effective for increasing therate of survival of dopaminergic neurons in the animal. In preferredembodiments, the method includes administering to the animal an amountof a subject compound that would otherwise be effective at protectingthe substantia nigra from MPTP-mediated toxicity when MPTP isadministered at a dose of 05 mg/kg, more preferably at a dose of 2mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg or 50 mg/kg and, still morepreferably, at a dose of 100 g/kg.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative diseasein which the loss of spinal and cranial motoneurons is a definingfeature. Treatment of patients suffering from such degenerativeconditions can include the application of a subject compound in order tocontrol, for example, apoptotic events that give rise to loss ofmotoneurons (e.g., to enhance survival of existing neurons).

Recently it has been reported that in certain ALS patients and animalmodels a significant loss of midbrain dopaminergic neurons occurs inaddition to the loss of spinal motor neurons. For instance, theliterature describes degeneration of the substantia nigra in somepatients with familial amyotrophic lateral sclerosis (Kostic et al. AnnNeurol 1997, 41, 497-504). According the subject invention, a trophicamount of a subject compound can be administered to an animal sufferingfrom, or at risk of developing, ALS.

In general, the therapeutic method of the present invention can becharacterized as including a step of administering to an animal anamount of a subject compound effective to enhance the survival of adopaminergic neurons and/or motoneurons. The mode of administration anddosage regimens will vary depending on the severity of the degenerativedisorder being treated, e.g., the dosage may be altered as between aprophylaxsis and treatment. In preferred embodiments, the subjectcompound is administered systemically initially, then locally formedium- to long-term care. In certain embodiments, a source of a subjectcompound is stereotactically provided within or proximate the area ofdegeneration. The subject method may also find particular utility intreating or preventing the adverse neurological consequences of surgery.For example, certain cranial surgery can result in degeneration ofneuronal populations for which the subject method can be applied.

In other embodiments, the subject method can be used to prevent or treatneurodegenerative conditions arising from the use of certain drugs, suchas the compound MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine).

In still other embodiments, the subject method can be used in theprevention and/or treatment of hypoxia, e.g., as a neuroprotectiveagent. For instance, the subject method can be used prophylactically tolessen the neuronal cell death caused by altitude-induced hypoxia.

A method which is “neuroprotective”, in the case of dopaminergic neuronsand motoneurons, results in diminished loss of cells of those phenotyperelative to that which would occur in the absence of treatment with asubject compound.

The subject method further has wide applicability to the treatment orprophylaxis of disorders affecting the regulation of peripheral nerves,including peripheral ganglionic neurons, sympathetic, sensory neurons,and motoneurons. In general, the method can be characterized asincluding administering to an animal an amount of a subject compoundeffective to alter the proliferative and/or differentiation state oftreated peripheral nerve cells. Such therapeutic compositions may beuseful in treatments designed to rescue, for example, retinal ganglia,inner ear and acoustic nerves, and motor neurons, from lesion-induceddeath as well as guiding reprojection of these neurons after suchdamage. Such diseases and conditions include, but are not limited to,chemical or mechanical trauma, infection (such as viral infection withvaricella zoster), metabolic disease such as diabetes, nutritionaldeficiency, and toxic agents (such as cisplatin used in the treatment ofa tumor). The goals of treatment in each case can be twofold: (1) toeliminate the cause of the disease and (2) to relieve its symptoms.

Peripheral neuropathy is a condition involving nerve-ending damage inthe hands and feet. Peripheral neuropathy generally refers to a disorderthat affects the peripheral nerves, most often manifested as one or acombination of motor, sensory, sensorimotor, or autonomic neuraldysfunction. The wide variety of morphologies exhibited by peripheralneuropathies can each be uniquely attributed to an equally wide varietyof causes. For instance, peripheral neuropathies can be geneticallyacquired, can result from a systemic disease, or can be induced by atoxic agent. Some toxic agents that cause neurotoxicities aretherapeutic drugs, antineoplastic agents, contaminants in foods ormedicinals, and environmental and industrial pollutants.

Peripheral neuropathy is a term used to describe disorders resultingfrom injury (e.g., mechanical, chemical, viral, bacterial, or genetic)to the peripheral nerves. It can be caused by diseases that affect onlythe peripheral nerves or by conditions that affect other parts of thebody as well. Symptoms almost always involve weakness, numbness, orpain—usually in the arms and legs.

To further illustrate, the subject method can be used in the treatmentof such acquired neuropathies as diabetic neuropathies; immune-mediatedneuropathies such as Guillain-Barre syndrome (GBS) and variants, chronicinflammatory demyelinating polyneuropathy (CIDP), chronicpolyneuropathies with antibodies to peripheral nerves, neuropathiesassociated with vasculitis or inflammation of the blood vessels inperipheral nerve, brachial or lumbosacral plexitis, and neuropathiesassociated with monoclonal gammopathies; neuropathies associated withtumors or neoplasms such as sensory neuropathy associated with lungcancer, neuropathy associated with multiple myeloma, neuropathyassociated with waldenstrom's macroglobulemia, chronic lymphocyticleukemia, or B-cell lymphoma; neuropathy associated with amyloidosis;neuropathies caused by infections; neuropathies caused by nutritionalimbalance; neuropathy in kidney disease; hypothyroid neuropathy;neuropathy caused by alcohol and toxins; neuropathies caused by drugs;neuropathy resulting from local irradiation; neuropathies caused bytrauma or compression; and idiopathic neuropathies.

Likewise, the subject method can be used in the treatment of suchhereditary neuropathies as Charcot-Marie-Tooth disease (CMT); familialamyloidotic neuropathy and other hereditary neuropathies; and hereditaryporphyria.

In another embodiment, the subject method can be used to inhibit orotherwise slow neurodegenerative events associated with age-relatedneuropathology.

In particular, chemotherapeutic agents known to cause sensory and/ormotor neuropathies include vincristine, an antineoplastic drug used totreat haematological malignancies and sarcomas, as well as cisplatin,taxol, and others. The neurotoxicity is dose-related, and exhibits asreduced intestinal motility and peripheral neuropathy, especially in thedistal muscles of the hands and feet, postural hypotension, and atony ofthe urinary bladder. Similar problems have been documented with taxoland cisplatin (Mollman, J. E., New Eng Jour Med 1990, 322, 126-127),although cisplatin-related neurotoxicity can be alleviated with nervegrowth factor (NGF) (Apfel, S. C. et al, Annals of Neurology 1992, 31,76-80). Although the neurotoxicity is sometimes reversible after removalof the neurotoxic agent, recovery can be a very slow process (Legha, S.,Medical Toxicology 1986, 1, 421-427; Olesen, et al., Drug Safety 1991,6, 302-314).

There are a number of inherited peripheral neuropathies, including:Refsum's disease, Abetalipoproteinemia, Tangier disease, Krabbe'sdisease, metachromatic leukodystrophy, Fabry's disease, Dejerine-Sottassyndrome, and others. Of all the inherited neuropathies, the most commonby far is Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth (CMT) Disease (also known as peroneal muscularatrophy, or hereditary motor sensory neuropathy (HMSN)) is the mostcommon hereditary neurological disorder. It is characterized by weaknessand atrophy, primarily of the peroneal muscles, due to segmentaldemyelination of peripheral nerves and associated degeneration of axonsand anterior horn cells. Autosomal dominant inheritance is usual, andassociated degenerative CNS disorders, such as Friedreich's ataxia, arecommon.

In one aspect, the method of the present invention can be used in thetreatment and maintenance of hereditary neuropathies. This group ofneuropathies is now becoming increasingly recognized due to the dramaticadvances in molecular genetics. The symptoms of the various hereditaryneuropathies are wide ranging. A common denominator is usually the earlyonset of mild numbness and tingling in the feet that slowly progressesto involve the legs and the hands and later the rest of the upperextremities. Most of the hereditary neuropathies do have a motorcomponent consisting of distal weakness in the lower and upperextremities. A majority of patients with hereditary neuropathies havehigh arches in their feet or other bony deformities. The symptoms arevery slowly progressive and the majority of the patients are stillwalking two decades after the onset of their symptoms.

The diagnosis of a hereditary neuropathy is usually suggested with theearly onset of neuropathic symptoms, especially when a positive familyhistory is also present. Prior to the recent genetic advances, thediagnosis was supported by typical findings of marked slowing of thenerve conduction studies on electromyography and a nerve biopsy. Typicalfindings on a nerve biopsy include the presence of so-calledonion-bulbs, indicating a recurring demyelinating and remyelinating ofthe nerve fibers. With the most recent genetic advances, two majorhereditary neuropathies known as “Charcot-Marie-Tooth disease” and“hereditary neuropathy with liability to pressure palsies” can bediagnosed with a simple blood test that identifies the differentmutations responsible for these two entities.

Hereditary neuropathies are caused by genetic abnormalities transmittedfrom generation to generation. For several of these, the genetic defectis known, and tests are available for diagnosis and prenatal counseling.

As set forth above, the subject method can be used as part of atherapeutic regimen in the treatment of Charcot-Marie Tooth Disease(CMT). This is a general term given to the hereditary sensorimotorneuropathies. CMT type 1 (CMT 1) is associated with demyelination orbreakdown of the myelin sheaths. Several different abnormalities havebeen identified. CMT Type 1A is most commonly caused by duplication of agene encoding a myelin protein called PMP-22, and CMT type 1B is causedby a mutation in a myelin protein called the Po glycoprotein. CMTX is ahereditary sensorimotor neuropathy that affects only men. It is causedby a mutation in a gene encoding a protein called Connexin 32 on theX-chromosome.

In certain embodiments, the subject method can be used to treat, or atleast reduce the severity of, amyotrophic lateral sclerosis (ALS).According the subject invention, a trophic amount of a subject compoundcan be administered to an animal suffering from, or at risk ofdeveloping, ALS.

In another embodiment, the subject method can be used in the treatmentof familial amyloidotic neuropathy and other related hereditaryneuropathies. Amyloidotic neuropathy usually presents with pain, sensoryloss and autonomic dysfunction. It is caused by a mutation in a proteincalled transthyretin, resulting in deposition of the protein as amyloidin the peripheral nerves.

The subject method can be used in the treatment of hereditary porphyria,which can have components of peripheral neuropathy.

Still another hereditary neuropathy for which the subject methods can beused for treatment is hereditary sensory neuropathy Type II (HSN II).

The methods and compositions of the present invention can also be usedin the treatment and maintenance of acquired neuropathies.

For example, subject compounds can be used to prevent diabeticneuropathies. Diabetes is the most common known cause of neuropathy. Itproduces symptoms in approximately 10% of people with diabetes. In mostcases, the neuropathy is predominantly sensory, with pain and sensoryloss in the hands and feet. But some diabetics have mononeuritis ormononeuritis multiplex, which causes weakness in one or more nerves, orlumbosacral plexopathy or amyotrophy, which causes weakness in the legs.

The instant method can also be used in the treatment of immune-mediatedneuropathies. The main function of the immune system is to protect thebody against infectious organisms which enter from outside. In somecases, however the immune system turns against the body and causesautoimmune disease. The immune system consists of several types of whiteblood cells, including T-lymphocytes, which also regulate the immuneresponse; and B-lymphocytes or plasma cells, which secrete specializedproteins called antibodies. Sometimes, for unknown reasons, the immunesystem mistakenly attacks parts of the body such as the peripheralnenes. This is autoimmune peripheral neuropathy. There are severaldifferent types, depending on the part of the peripheral nerve that isattacked and the type of the immune reaction. The following are briefdescriptions of the neuropathies that are mediated by the immune system.

For instance, a subject compound can be used to treat Guillain-BarreSyndrome (GBS), an acute neuropathy because it comes on suddenly orrapidly. Guillain-Barre Syndrome can progress to paralysis andrespiratory failure within days or weeks after onset. The neuropathy iscaused when the immune system destroys the myelin sheaths of the motorand sensory nerves. It is often preceded by infection, vaccination ortrauma, and that is thought to be what triggers the autoimmune reaction.The disease is self-limiting, with spontaneous recovery within six toeight weeks. But the recovery is often incomplete.

Other neuropathies which begin acutely, and which can be treated by themethod of the present invention, include acute motor neuropathy, acutesensory neuropathy, and acute autonomic neuropathy, in which there is animmune attack against the motor, sensory or autonomic nerves,respectively. The Miller-Fisher syndrome is another variant in whichthere is paralysis of eye gaze, incoordination, and unsteady gait.

Still another acquired neuropathy which is may be treated by the subjectmethod is chronic inflammatory demyelinating polyneuropathy (CIDP). CIDPis thought to be a chronic and more indolent form of the Guillain-Barresyndrome. The disease progresses either with repeated attacks, calledrelapses, or in a stepwise or steady fashion. As in GBS, there appearsto be destruction of the myelin sheath by antibodies and T-lymphocytes.But since there is no specific test for CIDP, the diagnosis is based onthe clinical and laboratory characteristics.

Chronic polyneuropathies with antibodies to peripheral nerves is stillanother peripheral neuropathy for which the subject methods can beemployed to treat or prevent. In some types of chronic neuropathies,antibodies to specific components of nerve have been identified. Theseinclude demyelinating neuropathy associated with antibodies to themyelin-associated glycoprotein (MAG), motor neuropathy associated withantibodies to the gangliosides GM1 or GD1a, and sensory neuropathyassociated with anti-sulfatide or GD1b ganglioside antibodies. Theantibodies in these cases bind to oligosaccharide or sugar likemolecules, which are linked to proteins (glycoproteins) or lipids(glycolipids or gangliosides) in the nerves. It is suspected that theseantibodies may be responsible for the neuropathies.

The subject method can also be used as part of a therapeutic plan fortreating neuropathies associated with vasculitis or inflammation of theblood vessels in peripheral nerves. Neuropathy can also be caused byvasculitis—an inflammation of the blood vessels in peripheral nerve. Itproduces small “strokes” along the course of the peripheral nerves, andmay be restricted to the nerves or it may be generalized, include a skinrash, or involve other organs. Several rheumatological diseases likerheumatoid arthritis, lupus, periarteritis nodosa, or Sjogren'ssyndrome, are associated with generalized vasculitis, which can alsoinvolve the peripheral nerves. Vasculitis can cause polyneuritis,mononeuritis, or mononeuritis multiplex, depending on the distributionand severity of the lesions.

In still another embodiment, the method of the present invention can beused for treatment of brachial or lumbosacral plexitis. The brachialplexus, which lies under the armpit, contains the nerves to the arm andhand. Brachial plexitis is the result of inflamation of that nervebundle, and produces weakness and pain in one or both arms. Lumbosacralplexitis, which occurs in the pelvis, causes weakness and pain in thelegs.

Subject compounds may also be suitable for use in the treatment ofneuropathies associated with monoclonal gammopathies. In monoclonalgammopathy, single clones of B-cells or plasma cells in the bone marrowor lymphoid organs expand to form benign or malignant tumors and secreteantibodies. The term ‘monoclonal’ is used because there are singleclones of antibodies, and ‘gammopathy’ stands for gammaglobulins, whichis another name for antibodies. In some cases, the antibodies react withnerve components; in others, fragments of the antibodies form amyloiddeposits.

Yet another aspect of the present invention relates to the use of thesubject method in the treatment of neuropathies associated with tumorsor neoplasms. Neuropathy can be due to direct infiltration of nerves bytumor cells or to indirect effect of the tumor. The latter is calledparaneoplastic neuropathy. Several types have been described. Forinstance, the subject methods can be used to manage sensory neuropathyassociated with lung cancer. This neuropathy is associated withantibodies to a protein called Hu, which is present in the sensoryneurons of the peripheral nerves. Likewise, the subject method can beused to treat neuropathies associated with multiple myeloma. Multiplemyeloma is a bony tumor that is caused by antibody-secreting plasmacells in the bone marrow. The tumor is made up of a single clone ofplasma cells, and the antibodies they produce are identical ormonoclonal. Some people with multiple myeloma develop a sensorimotorpolyneuropathy with degeneration of axons in the peripheral nerves. Inother embodiments, the subject method can be used to treat neuropathiesassociated with Waldenstrom's macroglobulemia, chronic lymphocyticleukemia, or B-cell lymphoma. These are tumors caused byantibody-secreting B-lymphocytes in the spleen, bone marrow or lymphnodes. These antibodies are monoclonal and frequently react withperipheral nerve components such as MAG, GM1, or sulfatide. In stillother embodiments, the compounds of the present invention can be used aspart of therapeutic protocol for the treatment of patients with cancerswhere neuropathy is a consequence of local irradiation or be caused bymedications such as vincristine and cisplatinum.

The present invention also contemplates the use of subject compounds forthe treatment of neuropathies associated with amyloidosis. Amyloid is asubstance that is deposited in the peripheral nerves and interferes withtheir operation: the resultant disorder is amyloidosis. There are twomain types: primary amyloidosis, in which the deposits contain fragmentsof monoclonal antibodies (see the monoclonal gammopathy paragraphabove); and hereditary amyloidosis in which the deposits contain amutated protein called transthyretin. Primary amyloidosis is usuallyassociated with monoclonal gammopathies or myeloma (see above).

Still another aspect of the present invention provides the subjectmethod as a means for treating neuropathies caused by infections.Peripheral neuropathies can be caused by infection of the peripheralnerves. Viruses that cause peripheral neuropathies include the AIDSvirus, HIV-I, which causes slowly progressive sensory neuropathy,cytomegalovirus, which causes a rapidly progressive paralyticneuropathy, Herpes zoster, which causes shingles, and poliovirus, whichcauses a motor neuropathy. Hepatitis B or C infections are sometimesassociated with vasculitic neuropathy.

Bacterial infections that cause neuropathy include leprosy, which causesa patchy sensory neuropathy, and diphtheria, which can cause a rapidlyprogressive paralytic neuropathy. Other infectious diseases that causeneuropathy include Lyme disease, which is caused by a spirochete, andtrypanosomiasis, which is caused by a parasite. Both commonly present amultifocal neuropathy.

Neuropathies caused by nutritional imbalance are also candidatedisorders for treatment by the subject method. Deficiencies of vitaminsB12, B1 (thiamine), B6 (pyridoxine), or E, for example, can producepolyneuropathies with degeneration of peripheral nerve axons. This canbe due to poor diet, or inability to absorb the nutrients from thestomach or gut.

Moreover megadoses of vitamin B6 can also cause a peripheral neuropathy,and the subject method can be used as part of a de-toxification programin such cases.

Yet another use of the subject method is in the treatment ofneuropathies arising in kidney diseases. Chronic renal failure can causea predominantly sensory peripheral neuropathy with degeneration ofperipheral nerve axons.

Another aspect of the present invention provides a method for treatinghypothyroid neuropathies. Hypothyroidism is sometimes associated with apainful sensory polyneuropathy with axonal degeneration. Mononeuropathyor mononeuropathy multiplex can also occur due to compression of theperipheral nerves by swollen tissues.

The subject method can also be used in the treatment of neuropathiescaused by alcohol and toxins. Certain toxins can cause peripheralneuropathy. Lead toxicity is associated with a motor neuropathy. Arsenicand mercury cause a sensory neuropathy. Thallium can cause a sensory andautonomic neuropathy. Several organic solvents and insecticides can alsocause polyneuropathy. Alcohol is directly toxic to nerves and alcoholabuse is a major cause of neuropathy. The subject method can be used, incertain embodiments, as part of a broader detoxification program.

In still another embodiment, the methods and compositions of the presentinvention can be used for the treatment of neuropathies caused by drugs.Several drugs are known to cause neuropathy. They include, among others,vincristine and cisplatinum in cancer, nitrofurantoin, which is used inpyelonephritis, amiodarone in cardiac arrhythmias, disulfuram inalcoholism, ddC and ddI in AIDS, and dapsone which is used to treatleprosy. As above, the subject method can be used, in certainembodiments, as part of a broader detoxification program.

The method of the present invention can also be used in the treatment ofneuropathies caused by trauma or compression. Localized neuropathies canresult from compression of nerves by external pressure or overlyingtendons and other tissues. The best known of these are the carpal tunnelsyndrome which results from compression at the wrist, and cervical orlumbar radiculopathies (sciatica), which result from compression ofnerve roots as they exit the spine. Other common areas of nervecompression include the elbows, armpits, and the back of the knees.

The subject method is also useful in variety of idiopathic neuropathies.The term “idiopathic” is used whenever the cause of the neuropathycannot be found. In these cases, the neuropathy is classified accordingto its manifestations, i.e., sensory, motor, or sensorimotor idiopathicpolyneuropathy.

Another aspect of the invention provides a conjoint therapy wherein oneor more other therapeutic agents are administered with the subjectcompound. Such conjoint treatment may be achieved by way of thesimultaneous, sequential or separate dosing of the individual componentsof the treatment. Conjoint administration thus includes administrationas part of the same pharmaceutical preparation, simultaneousadministration of separate pharmaceutical preparations, as well asadministration of separate pharmaceutical preparations at differenttimes on the same day, adjacent days, or otherwise as part of a singletherapeutic regimen. For example, the subject method can be carried outconjointly with other neuroprotective agents. The dosages recited hereinwould be adjusted to compensate for such additional components in thetherapeutic composition. Progress of the treated patient can bemonitored by conventional methods.

In yet other embodiments, the subject method can be carried outconjointly with the administration of growth and/or trophic factors. Forinstance, the combinatorial therapy can include a trophic factor such asglial cell line-derived neurotrophic factor, nerve growth factor,cilliary neurotrophic factor, schwanoma-derived growth factor, glialgrowth factor, striatal-derived neuronotrophic factor, platelet-derivedgrowth factor, brain-derived neurotrophic factor (BDNF), and scatterfactor (HGF-SF). Antimitogenic agents can also be used, as for example,cytosine, arabinoside, 5-fluorouracil, hydroxyurea, and methotrexate.

Determination of a therapeutically effective amount and/or aprophylactically effective amount of a subject compound, e.g., to beadequately neuroprotective, can be readily made by the physician orveterinarian (the “attending clinician”), as one skilled in the art, bythe use of known techniques and by observing results obtained underanalogous circumstances. The dosages may be varied depending upon therequirements of the patient in the judgment of the attending clinician,the severity of the condition being treated, the risk of furtherdegeneration to the CNS, and the particular agent being employed. Indetermining the therapeutically effective trophic amount or dose, and/orthe prophylactically effective amount or dose, a number of factors areconsidered by the attending clinician, including, but not limited to:the specific cause of the degenerative state and its likelihood ofrecurring or worsening; pharmacodynamic characteristics of theparticular agent and its mode and route of administration; the desiredtime course of treatment; the species of mammal; its size, age, andgeneral health; the response of the individual patient; the particularcompound administered; the bioavailability characteristics of thepreparation administered; the dose regimen selected; the kind ofconcurrent treatment (i.e., the interaction of the subject compound withother co-administered therapeutics); and other relevant circumstances.

Treatment can be initiated with smaller dosages that are less than theoptimum dose of the agent. Thereafter, the dosage should be increased bysmall increments until the optimum effect under the circumstances isreached. For convenience, the total daily dosage may be divided andadministered in portions during the day if desired. A therapeuticallyeffective trophic amount and a prophylactically effectiveneuroprotective amount of a subject compound, for instance, is expectedto vary from concentrations about 0.1 nanogram per kilogram of bodyweight per day (ng/kg/day) to about 100 mg/kg/day.

Compounds which are determined to be effective for the prevention ortreatment of degeneration of dopaminergic neurons and motoneurons andthe like in animals, e.g., dogs, rodents, may also be useful intreatment of disorders in humans. Those skilled in the art of treatingin such disorders in humans will be guided, from the data obtained inanimal studies, to the correct dosage and route of administration of thecompound to humans. In general, the determination of dosage and route ofadministration in humans is expected to be similar to that used todetermine administration in animals.

The identification of those patients who are in need of prophylactictreatment for disorders marked by degeneration of dopaminergic neuronsand/or motoneurons is well within the ability and knowledge of oneskilled in the art. Certain of the methods for identification ofpatients that are at risk and that can be treated by the subject methodare appreciated in the medical arts, such as family history of thedevelopment of a particular disease state and the presence of riskfactors associated with the development of that disease state in thesubject patient. A clinician skilled in the art can readily identifysuch candidate patients, by the use of, for example, clinical tests,physical examination and medical/family history.

IV. Exemplary Compounds

Compounds suitable for use in the various compositions and methods ofthe invention include compounds having a structure represented ingeneral Formula I:

wherein, as valence and stability permit,

-   -   M represents, independently for each occurrence, a heteroatom or        a substituted or unsubstituted methylene group, such as —CH₂—,        —CHF—, —CHOH—, —CH(Me)—, —C(═O)—, etc., or two M taken together        represent substituted or unsubstituted ethene or ethyne;    -   R, independently for each occurrence, represents H or lower        alkyl, preferably H;    -   T and V, independently, are absent or represent —N(R)—, —O—,        —S—, or —Se—, preferably N(R), O, or S, most preferably N(R);    -   U and Y, independently, represent —C(═O)—, —C(═S)—, —S(O₂)—,        —S(O)—, or a methylene group optionally substituted with 1-2        C1-C2 lower alkyl groups, preferably —C(═O)—, —C(═S)—, or        —S(O₂)—;    -   X and Z, independently, are absent or represent —N(R)—, —O—,        —S—, or —Se—, preferably N(R), O, or S, most preferably N(R);    -   R₁ represents a substituted or unsubstituted alkyl, heteroalkyl,        carbocyclic aliphatic, heterocyclic aliphatic, aryl, or        heteroaryl group;    -   Ar represents a substituted or unsubstituted aryl or heteroaryl        group;    -   Ht represents a substituted or unsubstituted heterocyclic        aliphatic or heteroaryl group, preferably having a nitrogen        member atom; and    -   i represents an integer from 0 to 4, preferably from 0 to 2.

Certain compounds of Formula I suitable for use in the variouscompositions and methods of the invention have a structure representedin general Formula II:

wherein, as valence and stability permit,

-   -   M represents, independently for each occurrence, a heteroatom or        a substituted or unsubstituted methylene group, such as —CH₂—,        —CHF—, —CHOH—, —CH(Me)—, —C(═O)—, etc., or two M taken together        represent substituted or unsubstituted ethene or ethyne;    -   R, independently for each occurrence, represents H or lower        alkyl, preferably H;    -   T and V, independently, are absent or represent —N(R)—, —O—,        —S—, or —Se—, preferably N(R), O, or S, most preferably N(R);    -   U and Y, independently, represent —C(═O)—, —C(═S)—, —S(O₂)—,        —S(O)—, or a methylene group optionally substituted with 1-2        C1-C2 lower alkyl groups, preferably —C(═O)—, —C(═S)—, or        —S(O₂)—;    -   X and Z, independently, are absent or represent —N(R)—, —O—,        —S—, or —Se—, preferably N(R), O, or S, most preferably N(R);    -   Cy represents a substituted or unsubstituted carbocyclic        aliphatic, heterocyclic aliphatic, aryl, or heteroaryl group;    -   Ar represents a substituted or unsubstituted aryl or heteroaryl        group;    -   Ht represents a substituted or unsubstituted heterocyclic        aliphatic or heteroaryl group, preferably having a nitrogen        member atom; and    -   i represents an integer from 0 to 4, preferably from 0 to 2.

In certain embodiments of the above formulae, each of TUV and XYZ,independently, comprises an amide, ester, or sulfonamide linkage. Incertain such embodiments, TUV comprises an amide (e.g., T is absent, Urepresents a carbonyl, and V represents N(R)), and XYZ comprises asulfonamide (e.g., X is absent, Y represents a sulfonyl, and Zrepresents N(R)). In certain embodiments, R is H for all occurrences. Incertain embodiments, X and Z are absent, and Ht represents aheterocyclic aliphatic ring, such as a piperidino, morpholino, orpiperazinyl ring, e.g., forming an amide or sulfonamide with Y.

In certain embodiments of the above formulae, Cy represents asubstituted or unsubstituted aryl or heteroaryl group, preferably amonocyclic group. In certain embodiments, substituents on Cy areselected from halogen, lower alkyl, lower heteroalkyl, azide, lowerhaloalkyl, hydroxyl, lower acyl, amine, amide, cyano, nitro, azido,sulfhydryl, sulfate, sulfonate, sulfamoyl, and sulfonamido, preferablyhalogen, lower alkyl, lower heteroalkyl, lower haloalkyl, hydroxyl,lower acyl, amine, amide, cyano, and nitro.

In certain embodiments, Ar is a phenyl ring, wherein V and X arepreferably disposed in a para relationship. In certain embodiments, Aris otherwise unsubstituted, while in other embodiments, additionalsubstituents on Ar are selected from amino, hydroxyl, halogen, loweralkyl, and lower heteroalkyl.

In certain embodiments, Ht represents a nitrogen-containing heteroarylring, such as oxazole, thiazole, imidazole, or pyridyl, preferablyoxazole or thiazole.

In certain embodiments, M_(i) is absent (i.e., i=0) or represents aheteroalkyl or alkyl chain having from 1-3 member atoms.

In certain embodiments, the present invention contemplates the use ofcompounds having a structure represented in Formula III:

wherein, as valence and stability permit,

-   -   L is absent or represents C1-C3 alkyl or heteroalkyl;    -   V represents N(R), O, or S, preferably N(R);    -   U and Y, independently, represent —C(═O)—, —C(═S)—, —S(O₂)—,        —S(O)—, or a methylene group optionally substituted with 1-2        C1-C2 lower alkyl groups, preferably —C(═O)—, —C(═S)—, or        —S(O₂)—;    -   W represents NH, O, or S, preferably O or S;    -   Z represents —N(R)—, —O—, —S—, or —Se—, preferably N(R), O, or        S, most preferably N(R);    -   Cy represents a substituted or unsubstituted carbocyclic        aliphatic, heterocyclic aliphatic, aryl, or heteroaryl group;        and    -   R₁ is absent or represents from 1 to 4 substituents on the ring        to which it is attached, such as halogen, alkyl, alkenyl,        alkynyl, azide, haloalkyl, hydroxyl, carbonyl (such as carboxyl,        alkoxycarbonyl, formyl, ketone, or acyl), thiocarbonyl (such as        thioester, thioacetate, or thioformate), alkoxyl, phosphoryl,        phosphonate, phosphinate, amine, amide, amidine, imine, cyano,        nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate,        sulfamoyl, sulfonamido, sulfonyl, silyl, ether, cycloalkyl,        heterocyclyl, heteroalkyl, heteroalkenyl, and heteroalkynyl,        heteroaralkyl, aralkyl, aryl or heteroaryl; and    -   R₂ is absent or represents from 1 to 2 substituents on the ring        to which it is attached, selected from halogen, lower alkyl,        lower haloalkyl, hydroxyl, formyl, acetyl, thiocarbonyl, lower        alkoxyl, amine, amide, cyano, nitro, lower alkylthio, sulfate,        and sulfamoyl, preferably from halogen, lower alkyl, lower        haloalkyl, hydroxyl, acetyl, methoxy, amine, and cyano, most        preferably from halogen, lower alkyl, and lower haloalkyl.

In certain embodiments, R₁ is selected from halogen, hydroxyl, amino,lower alkyl, and lower heteroalkyl. In certain embodiments, R₁ isabsent.

In certain embodiments, R₂ is selected from halogen, hydroxyl, amino,lower alkyl, and lower heteroalkyl. In certain embodiments, R₂ isabsent.

In certain embodiments, UV taken together represent amine or thioamide,preferably amide, and YZ taken together represent sulfonamide.

In certain embodiments, Cy represents a substituted or unsubstitutedaryl or heteroaryl group, preferably a monocyclic group. In certainembodiments, substituents on Cy are selected from halogen, lower alkyl,lower heteroalkyl, azide, lower haloalkyl, hydroxyl, lower acyl, amine,amide, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl,and sulfonamido, preferably halogen, lower alkyl, lower heteroalkyl,lower haloalkyl, hydroxyl, lower acyl, amine, amide, cyano, and nitro.

Certain compounds of Formula I suitable for use in the variouscompositions and methods of the invention have a structure representedin general Formula IV:

wherein, as valence and stability permit,

-   -   M represents, independently for each occurrence, a heteroatom or        a substituted or unsubstituted methylene group, such as —CH₂—,        —CHF—, —CHOH—, —CH(Me)—, —C(═O)—, etc., or two M taken together        represent substituted or unsubstituted ethene or ethyne;    -   Q represents, independently for each occurrence, O or S,        preferably O;    -   R, independently for each occurrence, represents H or lower        alkyl, preferably H;    -   T and V, independently, are absent or represent —N(R)—, —O—,        —S—, or —Se—, preferably N(R), O, or S, most preferably N(R);    -   U and Y, independently, represent —C(═O)—, —C(═S)—, —S(O₂)—,        —S(O)—, or a methylene group optionally substituted with 1-2        C1-C2 lower alkyl groups, preferably —C(═O)—, —C(═S)—, or        —S(O₂)—;    -   X and Z, independently, are absent or represent —N(R)—, —O—,        —S—, or —Se—, preferably N(R), O, or S, most preferably N(R);    -   Cy represents a substituted or unsubstituted carbocyclic        aliphatic, heterocyclic aliphatic, aryl, or heteroaryl group;    -   Ar represents a substituted or unsubstituted aryl or heteroaryl        group;    -   Ht represents a substituted or unsubstituted heterocyclic        aliphatic or heteroaryl group, preferably having a nitrogen        member atom; and    -   i represents an integer from 0 to 4, preferably from 0 to 2.

In certain embodiments, each of TUV and XYZ, independently, comprises anamide, ester, or sulfonamide linkage. In certain such embodiments, TUVcomprises an amide (e.g., T is absent, U represents a carbonyl, and Vrepresents N(R)), and XYZ comprises a sulfonamide (e.g., X is absent, Yrepresents a sulfonyl, and Z represents N(R)). In certain embodiments, Ris H for all occurrences. In certain embodiments, X and Z are absent,and Ht represents a heterocyclic aliphatic ring, such as a piperidino,morpholino, or piperazinyl ring, e.g., forming an amide or sulfonamidewith Y.

In certain embodiments, Cy represents a substituted or unsubstitutedaryl or heteroaryl group, preferably a monocyclic group. In certainembodiments, substituents on Cy are selected from halogen, lower alkyl,lower heteroalkyl, azide, lower haloalkyl, hydroxyl, lower acyl, amine,amide, cyano, nitro, azido, sulfhydryl, sulfate, sulfonate, sulfamoyl,and sulfonamido, preferably halogen, lower alkyl, lower heteroalkyl,lower haloalkyl, hydroxyl, lower acyl, amine, amide, cyano, and nitro.

In certain embodiments, Ar is a phenyl ring, wherein V and X arepreferably disposed in a para relationship. In certain embodiments, Aris otherwise unsubstituted, while in other embodiments, additionalsubstituents on Ar are selected from amino, hydroxyl, halogen, loweralkyl, and lower heteroalkyl.

In certain embodiments, Ht represents a nitrogen-containing heteroarylring, such as oxazole, thiazole, imidazole, or pyridyl, preferablyoxazole or thiazole.

In certain embodiments, M_(i) is absent (i.e., i=0) or represents aheteroalkyl or alkyl chain having from 1-3 member atoms.

Representative examples of compounds encompassed by the above formulaethat are useful in the compositions and methods of the present inventioninclude the following molecular structures, identified (ID) as compoundsA-D″″″ whose bioactivities are shown in Table 1:

V. Exemplary Assays

Cell-based functional assays can be employed for identifying additionalcompounds such as those disclosed herein which exhibit similar oranalogous therapeutic activity. Suitable assays are cited and describedin the Exemplification below.

Potential therapeutic compounds, such as described above, can be testedby any of number of well known animal disease models. For instance,regarding Parkinson's disease, selected agents can be evaluated inanimals treated with MPTP. The compound MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and its metabolite MPP⁺have been used to induce experimental parkinsonism. MPP⁺ killsdopaminergic neurons in the substantia nigra, yielding a reasonablemodel of late parkinsonism. Turski et al., (1991) Nature 349:414.

Other models of Parkinson's disease are known in the art, such as the6-hydroxydopamine model (6-OHDA) and the axotomy model. In the formermodel, a nigral cell body lesion is produced by injecting 6-OHDAunilaterally into the rat medial forebrain bundle. In the latter, nigralneurons were induced to degenerate by transecting their axons within themedial forebrain bundle. These models are described in Lin, PromegaNeural Notes 1996, 11, 3-7, and references cited therein.

VI. Exemplary Pharmaceutical Preparations

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The compounds of the inventionmay be formulated for administration in any convenient way for use inhuman or veterinary medicine. In certain embodiments, the compoundincluded in the pharmaceutical preparation may be active itself, or maybe a prodrug, e.g., capable of being converted to an active compound ina physiological setting.

Thus, another aspect of the present invention provides pharmaceuticallyacceptable compositions comprising a therapeutically effective amount ofone or more of the compounds described above, formulated together withone or more pharmaceutically acceptable carriers (additives) and/ordiluents. As described in detail below, the pharmaceutical compositionsof the present invention may be specially formulated for administrationin solid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intrathecal, intracerebroventricular,intramuscular, or intravenous injection as, for example, a sterilesolution or suspension, including administration using a minipump orother mechanical-assisted delivery, such as ALZET osmotic pumps thatcontinuously deliver agents at controlled rates; (3) topicalapplication, for example, as a cream, ointment or spray applied to theskin; or (4) intravaginally or intrarectally, for example, as a pessary,cream or foam. However, in certain embodiments the subject compounds maybe simply dissolved or suspended in sterile water. In certainembodiments, the pharmaceutical preparation is non-pyrogenic, i.e., doesnot elevate the body temperature of a patient.

The phrase ‘therapeutically effective amount’ as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animaland thereby blocking the biological consequences of that pathway in thetreated cells, at a reasonable benefit/risk ratio applicable to anymedical treatment.

The phrase ‘pharmaceutically acceptable’ is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase ‘pharmaceutically acceptable carrier’ as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject compoundsfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be ‘acceptable’ in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

As set out above, certain subject compounds may contain a basicfunctional group, such as amino or alkylamino, and are, thus, capable offorming pharmaceutically acceptable salts with pharmaceuticallyacceptable acids. The term ‘pharmaceutically acceptable salts’ in thisrespect, refers to the relatively non-toxic, inorganic and organic acidaddition salts of compounds of the present invention. These salts can beprepared in situ during the final isolation and purification of thecompounds of the invention, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed. Representativesalts include the hydrobromide, hydrochloride, sulfate, bisulfate,phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate,laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate,fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate,lactobionate, and laurylsulphonate salts and the like. (See, forexample, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term ‘pharmaceutically acceptable salts’ in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, forexample, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient that can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about1 percent to about ninety-nine percent of active ingredient, preferablyfrom about 5 percent to about 70 percent, most preferably from about 10percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in microencapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants that may berequired.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”,W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feedsand Feeding” 0 and B books, Corvallis, Ore., U.S.A., 1977).

VII. Synthetic Schemes and Identification of Active Compounds

a. Combinatorial Libraries

The compounds of the present invention, particularly libraries ofvariants having various representative classes of substituents, areamenable to combinatorial chemistry and other parallel synthesis schemes(see, for example, PCT WO 94/08051). The result is that large librariesof related compounds, e.g., a variegated library of compoundsrepresented above, can be screened rapidly in high throughput assays inorder to identify potential lead compounds, as well as to refine thespecificity, toxicity, and/or cytotoxic-kinetic profile of a leadcompound.

Simply for illustration, a combinatorial library for the purposes of thepresent invention is a mixture or set of chemically related compoundsthat may be screened together for a desired property. The preparation ofmany related compounds in a single reaction greatly reduces andsimplifies the number of screening processes that need to be carriedout. Screening for the appropriate physical properties can be done byconventional methods.

Diversity in the library can be created at a variety of differentlevels. For instance, the substrate aryl groups used in thecombinatorial reactions can be diverse in terms of the core aryl moiety,e.g., a variegation in terms of the ring structure, and/or can be variedwith respect to the other substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules such as the subjectcompounds. See, for example, Blondelle et al. (1995) Trends Anal. Chem.14:83; the Affymax U.S. Pat. Nos. 5,359,115 and 5,362,899: the EllmanU.S. Pat. No. 5,288,514: the Still et al. PCT publication WO 94/08051;the ArQule U.S. Pat. Nos. 5,736,412 and 5,712,171; Chen et al. (1994)JACS 116:2661: Kerr et al. (1993) JACS 115:252; PCT publicationsWO92/10092, WO93/09668 and WO91/07087; and the Lerner et al. PCTpublication WO93/20242). Accordingly, a variety of libraries on theorder of about 100 to 1,000,000 or more diversomers of the subjectcompound scan be synthesized and screened for particular activity orproperty.

In an exemplary embodiment, a library of candidate compounds diversomerscan be synthesized utilizing a scheme adapted to the techniquesdescribed in the Still et al. PCT publication WO 94/08051, e.g., beinglinked to a polymer bead by a hydrolyzable or photolyzable group,optionally located at one of the positions of the candidate compounds ora substituent of a synthetic intermediate. According to the Still et al.technique, the library is synthesized on a set of beads, each beadincluding a set of tags identifying the particular diversomer on thatbead.

The structures of the compounds useful in the present invention lendthemselves readily to efficient synthesis. The nature of the structuresof the subject compounds, as generally set forth above, allows the rapidcombinatorial assembly of such compounds. For example, as depictedbelow, solid phase routes can be employed to rapidly assemble a widevariety of structures of Formulae I and II for testing in the assaysdescribed herein. The structure of the subject compounds is well suitedfor such an approach, because the combinations TUV and XYZ, or subsetsthereof, can be readily attached using reactions such as those depictedin FIGS. 1-31. These reactions generally are quite mild and have beensuccessfully applied in combinatorial solid-phase synthesis schemes.Furthermore, the wide range of substrates and coupling partners suitableand available for these reactions permits the rapid assembly of large,diverse libraries of compounds for testing in assays as set forthherein. For certain schemes, and for certain substitutions on thevarious substituents of the subject compounds, one of skill in the artwill recognize the need for masking certain functional groups with asuitable protecting group. Such techniques are well known in the art andare easily applied to combinatorial synthesis schemes.

In addition to the coupling steps depicted below, additional steps maybe used to elaborate or functionalize the basic structural subunits,such as Ar, Cy, and Ht, while the structures are bound to the solidsupport. Furthermore, many variations on the above and related pathwayspermit the synthesis of widely diverse libraries of compounds that canbe tested as therapeutics similar or analogous to those disclosedherein, e.g., by forming a T—U bond or X—Y bond as a coupling step,rather than forming the U—V or Y—Z bonds to couple subunits. All ofthese permutations and variations will be understood by one of skill inthe art to be included in the scope of the present invention.

Exemplary reactions useful for generating compounds of Formulae I and IIfor use in the methods and compositions of the present invention areshown in FIGS. 1-31. The reaction conditions in the illustrated schemesof FIG. 1-31 are as follows:

-   1) R₁CH₂CN, NaNH₂, toluene

(Arzneim-Forsch, 1990, 40, 11, 1242)

-   2) H₂SO₄, H₂O, reflux

(Arzneim-Forsch, 1990, 40, 11, 1242)

-   3) H₂SO₄, EtOH, reflux

(Arzneim-Forsch, 1990, 40, 11, 1242)

-   4) NaOH, EtOH, reflux-   5) (Boc)₂O, 2M NaOH, THF-   6) LiHDMS, R₁X, THF

(Merck Patent Applic # WO 96/06609)

-   7) Pd—C, H₂, MeOH-   8) t-BuONO, CuBr, HBr, H₂O

(J. Org. Chem. 1977, 42, 2426)

-   9) ArB(OH)₂, Pd(PPh₃)₄, Dioxane

(J. Med. Chem. 1996, 39, 217-223)

-   10) R₁₂(H)C═CR₁₃R₁₄, Pd(OAc)₂, Et₃N, DMF

(Org. React. 1982, 27, 345)

-   11) Tf₂O, THF P (J. Am. Chem. Soc. 1987, 109, 5478-5486)-   12) ArSnBu₃, Pd(PPh₃)₄, Dioxane

(J. Am. Chem. Soc. 1987, 109, 5478-5486)

-   13) KMnO₄, Py, H₂O

(J. Med. Chem. 1996, 39, 217-223)

-   14) NaOR₁, THF-   15) NaSR₁, THF-   16) HNR₁R₁₃, THF-   17) HONO, NaBF₄

(Adv. Fluorine Chem. 1965, 4, 1-30)

-   18) Pd(OAc)₂, NaH, DPPF, PhCH₃, R₁₀H

(J. Org. Chem. 1997, 62, 5413-5418)

-   19) i. R₁X, Et₃N, CH₂Cl₂, ii. R₁₃X-   20) SOCl₂, cat DMF-   21) CH₂N₂, Et₂O-   22) Ag₂O, Na₂CO₃, Na₂S₂O₃, H₂O

(Tetrahedron Lett. 1979, 2667)

-   23) AgO₂CPh, Et₃N, MeOH

(Org. Syn., 1970, 50, 77; J. Am. Chem. Soc. 1987, 109, 5432)

-   24) LiOH, THF-MeOH-   25) (EtO)₂P(O)CH₂CO₂R, BuLi, THF-   26) MeO₂CCH(Br)═P(Ph)₃, benzene-   27) KOH or KOtBu-   28) Base, X(CH₂)_(n)CO₂R-   29) DPPA, Et₃N, toluene

(Synthesis 1985, 220)

-   30) HONO, H₂O-   31) SO₂, CuCl, HCl, H₂O

(Synthesis 1969, 1-10, 6)

-   32) Lawesson's reagent, toluene

(Tetrahedron Asym. 1996, 7, 12, 3553)

-   33) R₂M, solvent-   34) 30% H₂O₂, glacial CH₃CO₂H

(Helv. Chim. Acta. 1968, 349, 323)

-   35) triphosgene, CH₂Cl₂

(Tetrahedron Lett., 1996, 37, 8589)

-   36) i. (EtO)₂P(O)CHLiSO₂Oi-Pr, THF, ii. NaI-   37) Ph₃PCH₃I, NaCH₂S(O)CH₃, DMSO

(Synthesis 1987, 498)

-   38) Br₂, CHCl₃ or other solvent

(Synthesis 1987, 498)

-   39) BuLi, Bu₃SnCl-   40) ClSO₂OTMS, CCl₄

(Chem. Ber. 1995, 128, 575-580)

-   41) MeOH—HCl, reflux-   42) LAH, Et₂O or LiBH₄, EtOH or BH₃-THF (Tetrahedron Lett., 1996,    37, 8589)-   43) MsCl, Et₃N, CH₂Cl₂

(Tetrahedron Lett., 1996, 37, 8589)

-   44) Na₂SO₃, H₂O

(Tetrahedron Lett., 1996, 37, 8589)

-   45) R₂R₄NH, Et₃N, CH₂Cl₂-   46) R₂M, solvent-   47) CH₃NH(OCH₃), EDC, HOBt, DIEA, CH₂Cl₂ or DMF

(Tetrahedron Lett, 1981, 22, 3815)

-   48) MeLi, THF-   49) mCPBA, CH₂Cl₂-   50) HONO, Cu₂O, Cu(NO₃)₂, H₂O

(J. Org. Chem. 1977, 42, 2053)

-   51) RIM, solvent-   52) HONO, NaS(S)COEt, H₂O

(Org. Synth. 1947, 27, 81)

-   53) HSR₂ or HSR₄, CH₂Cl₂-   54) i-BuOC(O)Cl, Et₃N, NH₃, THF-   55) R₂R₄NH, CH₂Cl₂, NaBH(OAc)₃-   56) R₂R₄NH, MeOH/CH₃CO₂H, NaBH₃CN-   57) R₂OH, EDC, HOBt, DIEA, CH₂Cl₂ or DMF-   58) R₂OH, HBTU, HOBt, DIEA, CH₂Cl₂ or DMF-   59) R₂R₄NH, EDC, HOBt, DIEA, CH₂Cl₂ or DMF-   60) R₂R₄NH, HBTU, HOBt, DIEA, CH₂Cl₂ or DMF-   61) POCl₃, Py, CH₂Cl₂-   62) R₂R₄NCO, solvent-   63) R₂OC(O)Cl, Et₃N, solvent-   64) R₂CO₂H, EDC or HBTU, HOBt, DIEA, CH₂Cl₂ or DMF-   65) R₂X, Et₃N, solvent-   66) (CH₃S)₂C═N(CN), DMF, EtOH

(J. Med. Chem. 1994, 37, 57-66)

-   67) R₂SO₂Cl, Et₃N, CH₂Cl₂-   68) R₂— or R₃— or R₄—CHO, MeOH/CH₃CO₂H, NaBH₃CN

(Synthesis 1975, 135-146)

-   69) Boc(Tr)-D or L-CysOH, HBTU, HOBt, DIEA, CH₂Cl₂ or DMF-   70) Boc(Tr)-D or L-CysH, NaBH₃CN, MeOH/CH₃CO₂H

(Synthesis 1975, 135-146)

-   71) S-Tr-N-Boc cysteinal, ClCH₂CH₂Cl or THF, NaBH(OAc)₃

(J. Org. Chem. 1996, 61, 3849-3862)

-   72) TFA, CH₂Cl₂, Et₃ SiH or (3:1:1) thioanisole/ethanedithiol/DMS-   73) TFA, CH₂Cl₂-   74) DPPA, Et₃N, toluene, HOCH₂CH₂SiCH₃

(Tetrahedron Lett. 1984, 25, 3515)

-   75) TBAF, THF-   76) Base, TrSH or BnSH-   77) Base, R₂X or R₄X-   78) R₃NH₂, MeOH/CH₃CO₂H, NaBH₃CN-   79) N₂H₄, KOH-   80) Pd₂(dba)₃, P(o-tol)₃, RNH₂, NaOtBu, Dioxane, R₁NH₂

(Tetrahedron Lett. 1996, 37, 7181-7184).

-   81) Cyanamide.-   82) Fmoc-Cl, sodium bicarbonate.-   83) BnCOCl, sodium carbonate.-   84) AllylOCOCl, pyridine.-   85) Benzyl bromide, base.-   86) Oxalyl chloride, DMSO.-   87) RCONH₂.-   88) Carbonyldiimidazole, neutral solvents (e.g., DCM, DMF, THF,    toluene).-   89) Thiocarbonyldiimidazole, neutral solvents (e.g., DCM, DMF, THF,    toluene).-   90) Cyanogen bromide, neutral solvents (e.g., DCM, DMF, THF,    toluene).-   91) RCOCl, Triethylamine-   92) RNHNH₂, EDC.-   93) RO₂CCOCl, Et₃N, DCM.-   94) MsOH, Pyridine (J. Het. Chem., 1980, 607.)-   95) Base, neutral solvents (e.g., DCM, toluene, THF).-   96) H₂NOR, EDC.-   97) RCSNH₂.-   98) RCOCHBrR, neutral solvents (e.g., DCM, DMF, THF, toluene), (Org.    Proc. Prep. Intl., 1992, 24, 127).-   99) CH₂N², HCl. (Synthesis, 1993, 197).-   100) NH₂NHR, neutral solvents (e.g., DCM, DMF, THF, toluene).-   101) RSO₂Cl, DMAP. (Tetrahedron Lett., 1993, 34, 2749).-   102) Et₃N, RX. (J. Org. Chem., 1990, 55, 6037).-   103) NOCl or Cl₂ (J. Org. Chem., 1990, 55, 3916).-   104) H₂NOH, neutral solvents (e.g., DCM, DMF, THF, toluene).-   105) RCCR, neutral solvents (DCM, THF, Toluene).-   106) RCHCHR, neutral solvents (DCM, THF, Toluene).-   107) H₂NOH, HCl.-   108) Thiocarbonyldiimidazole, SiO₂ or BF₃OEt₂. (J. Med. Chem., 1996,    39, 5228).-   109) Thiocarbonyldiimidazole, DBU or DBN. (J. Med. Chem., 1996, 39,    5228).-   110) HNO₂, HCl.-   111) ClCH₂CO₂Et (Org. Reactions, 1959, 10, 143).-   112) Morpholine enamine (Eur. J. Med. Chem., 1982, 17, 27).-   113) RCOCHR′CN-   114) RCOCHR′CO₂Et-   115) Na₂SO₃-   116) H₂NCHRCO₂Et-   117) EtO₂CCHRNCO-   118) RCNHNH₂.-   119) RCOCO₂H, (J. Med. Chem., 1995, 38, 3741).-   120) RCHO, KOAc.-   121) 2-Fluoronitrobenzene.-   122) SnCl₂, EtOH, DMF.-   123) RCHO, NaBH₃CN, HOAc.-   124) NH₃, MeOH.-   125) 2,4,6-Me₃PhSO₂NH₂.-   126) Et₂NH, CH₂Cl₂-   127) MeOC(O)Cl, Et₃N, CH₂Cl₂-   128) R₂NH₂, EDC, HOBT, Et₃N, CH₂Cl₂-   129) DBU, PhCH₃-   130) BocNHCH(CH₂STr)CH₂NH₂, EDC, HOBT, Et₃N, CH₂Cl₂-   131) R₂NHCH₂CO₂Me, HBTU, HOBT, Et₃N, CH₂Cl₂-   132) BocNHCH(CH₂STr)CH₂OMs, LiHMDS, THF-   133) R₂NHCH₂CO₂Me, NaBH(OAc)₃, ClCH₂CH₂Cl or THF-   134) R₂NHCH₂CH(OEt)₂, HBTU, HOBT, Et₃N, CH₂Cl₂-   135) NaBH(OAC)₃, ClCH₂CH₂Cl or THF, AcOH.-   136) Piperidine, DMF.-   137) Pd(Ph₃P)₄, Bu₃SnH.-   138) RCO₂H, EDC, HOBT, Et₃N, DCM.-   139) RNH₂, neutral solvents.-   140) RCHO, NaBH₃CN, HOAc.-   141) RNCO, solvent.-   142) RCO₂H, EDC or HBTU, HOBt, DIEA, CH₂Cl₂ or DMF.-   143) RCOCl, Triethylamine-   144) RSO₂Cl, Et₃N, CH₂Cl₂.-   145) SnCl₂, EtOH, DMF.-   146) RNH₂, EDC, HOBt, DIEA, CH₂Cl₂ or DMF.-   147) Dibromoethane, Et₃N, CH₂Cl₂-   148) Oxalyl chloride, neutral solvents.-   149) LiOH, THF-MeOH.-   150) Carbonyldiimidazole, neutral solvents (e.g., DCM, DMF, THF,    toluene).-   151) RNH₂, Et₃N, CH₂Cl₂.-   152) Base, RX.-   153) DBU, PhCH₃-   154) DPPA, Et₃N, toluene (Synthesis 1985, 220)-   155) SOCl₂, cat DMF.-   156) ArH, Lewis Acid (AlCl₃, SnCl₄, TiCl₄), CH₂Cl₂.-   157) H₂NCHRCO₂Et, neutral solvents.-   158) BocHNCHRCO₂H, EDC OR HBTU, HOBt, DIEA, CH₂Cl₂ or DMF.-   159) TFA, CH₂Cl₂.

VIII. Business Methods

One aspect of the present invention relates to a kit comprisingcompounds as described herein, e.g., of Formula I, II, III or IV, forpromoting survival of substantia nigra neuronal cells, dopaminergiccells, or motoneurons, in a patient, preferably a human, and inassociation with instructions (written and/or pictorial) describing theuse of the formulation for promoting survival of substantia nigraneuronal cells, dopaminergic cells, or motoneurons, and optionally,warnings of possible side effects and drug-drug or drug-foodinteractions.

Another aspect of the present invention relates to a kit comprisingcompounds as described herein, e.g., of Formula I, II, III or IV, fortreating a disorder characterized by loss of dopaminergic neurons and/ormotoneurons, or for treating or preventing Parkinson's disease, or ALS,or limiting damage to neuronal cells by Parkinsonian conditions, in apatient, preferably a human, and in association with instructions(written and/or pictorial) describing the use of the formulation fortreating a disorder characterized by loss of dopaminergic neurons and/ormotoneurons, or for treating or preventing Parkinson's disease, or ALS,or limiting damage to neuronal cells by Parkinsonian conditions, andoptionally, warnings of possible side effects and drug-drug or drug-foodinteractions.

The invention further contemplates a method for conducting apharmaceutical business, comprising: (a) manufacturing a pharmaceuticalpreparation comprising a sterile pharmaceutical excipient and compoundsas described herein, e.g., of Formula II or II; and (b) marketing (e.g.,providing promotional and/or informative presentations (such asdisplays, telemarketing, and lectures), products (such as trial samplesof the preparation), and/or documentation (including leaflets,pamphlets, websites, posters, etc.)) to healthcare providers, such asdoctors, hospitals, clinics, etc., a benefit of using the pharmaceuticalpreparation for promoting survival of substantia nigra neuronal cells,dopaminergic cells, or motoneurons.

The invention further contemplates a method for conducting apharmaceutical business, comprising: (a) manufacturing a pharmaceuticalpreparation comprising a sterile pharmaceutical excipient and compoundsas described herein, e.g., of Formula I, II, III or IV; and (b)marketing (e.g., providing promotional and/or informative presentations(such as displays, telemarketing, and lectures), products (such as trialsamples of the preparation), and/or documentation (including leaflets,pamphlets, websites, posters, etc.)) to healthcare providers, such asdoctors, hospitals, clinics, etc., a benefit of using the pharmaceuticalpreparation for treating a disorder characterized by loss ofdopaminergic neurons and/or motoneurons, or for treating or preventingParkinson's disease, or ALS, or limiting damage to neuronal cells byParkinsonian conditions.

Another aspect of the invention provides for a method for conducting apharmaceutical business, comprising: (a) providing a distributionnetwork for selling the pharmaceutical composition comprising a sterilepharmaceutical excipient and compounds as described herein, e.g., ofFormula I, II, III or IV; and (b) providing instruction material topatients or physicians for using the pharmaceutical composition forpromoting survival of substantia nigra neuronal cells, dopaminergiccells, or motoneurons.

Another aspect of the invention provides for a method for conducting apharmaceutical business, comprising: (a) providing a distributionnetwork for selling the pharmaceutical composition comprising a sterilepharmaceutical excipient and compounds as described herein, e.g., ofFormula I, II, III or IV; and (b) providing instruction material topatients or physicians for treating a disorder characterized by loss ofdopaminergic neurons and/or motoneurons, or for treating or preventingParkinson's disease, or ALS, or limiting damage to neuronal cells byParkinsonian conditions.

Another aspect of the invention provides for a method for conducting apharmaceutical business, comprising: (a) determining an appropriatepharmaceutical preparation and dosage of a compounds as describedherein, e.g., of Formula I, II, III or IV, for promoting survival ofsubstantia nigra neuronal cells, dopaminergic cells, or motoneurons; (b)conducting therapeutic profiling of the pharmaceutical preparation forefficacy and toxicity in animals; (c) providing a distribution networkfor selling a pharmaceutical composition having an acceptabletherapeutic profile; and, optionally, (d) providing a sales group formarketing the preparation to healthcare providers.

Yet another aspect of the invention provides for a method for conductinga pharmaceutical business, comprising: (a) determining an appropriatepharmaceutical preparation and dosage of a compounds as describedherein, e.g., of Formula I, II, III or IV, for treating a disordercharacterized by loss of dopaminergic neurons and/or motoneurons, or fortreating or preventing Parkinson's disease, or ALS, or limiting damageto neuronal cells by Parkinsonian conditions; (b) conducting therapeuticprofiling of the pharmaceutical preparation for efficacy and toxicity inanimals; (c) providing a distribution network for selling apharmaceutical composition having an acceptable therapeutic profile;and, optionally, (d) providing a sales group for marketing thepreparation to healthcare providers.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Parkinson's disease (PD) is characterized by a dysfunction in thedopaminergic activity of the substantia nigra that is caused by neuronaldegeneration. This results in a state of dopamine (DA) deficiencycausing the movement disorder symptoms including rigidity, tremorbradykinesis, gait difficulty, and postural instability. The mosteffective treatment for PD to date is the oral administration of thedopamine precursor Levodopa. Levodopa penetrates the central nervoussystem and is enzymatically converted to dopamine. It is believed thatbeneficial effects of Levodopa result from increased concentration ofdopamine in the brain. Unfortunately, neither Levodopa nor any of theless commonly utilized medications typically stem the progression of thedisease, which is caused by the degeneration of dopaminergic neurons inthe substantia nigra

Neurotrophic activity promotes the survival and maintains the phenotypicdifferentiation of nerve cells. Hence, neurotrophic molecules may beuseful in protecting responsive neurons against a variety of differentforms of nerve damages. Neurotrophic molecules specific for midbrainsubstantia nigra dopaminergic neurons would be of clinical interestbecause those neurons play an important role in the regulation of motoractivity and because progressive degeneration of the substantia nigradopaminergic neurons is the hallmark of PD. Here, we describe a methodfor screening small molecule libraries for neurotrophic activity on theembryonic precursors of the substantia nigra dopaminergic neurons thatdegenerate in PD. This bioassay for identifying neurotrophic activitydirected to dopaminergic neurons that may be useful in treating PD isbased on an assay previously described (Lin et al, J. Neurochem 1994,63, 758-768) and implemented with modification in the present invention.This bioassay used dissociated cell cultures of embryonic midbrain,where high-affinity dopamine (DA) uptake and expression of tyrosinehydroxylase (TH), the rate-limiting enzyme in DA synthesis, can be usedas markers for dopaminergic neuron survival and differentiation. Wescreened various libraries of natural products and small molecules fortrophic activities specific for mesencephalon dopaminergic neurons.

Compound A as depicted above was discovered based on its ability topromote the functional activity and survival in cell culture ofdopaminergic nerve cells isolated from the rat embryo mesencephalon,These dopaminergic nerve cells are the embryonic precursor of thedopaminergic nerve cell in the adult substantia nigra that degenerate inPD. Therefore, compound A and related compounds may be useful inreducing the neuronal degeneration that causes the symptoms of PD.

Furthermore, compound A may be useful in treating other forms of damageto or improper function of dopaminergic nerve cells in human patientssuch as schizophrenia and other forms of psychosis. Current treatmentsof such conditions require drugs active at dopamine receptors,suggesting that improper function of the dopaminergic neurons enervatingthese receptor-bearing neuronal populations may be involved in thedisease process,

Other conditions that may be treated with compound A include those thatare caused or contributed to by death or decreased function ofdopaminergic neurons.

Methods Cell Culture

Primary midbrain cultures were prepared from rat embryo ventralmesencephalon as described previously by Lin et al (1994). In brief,rostral mesencephalic tegmentum of embryonic day 16 rat embryos wascollected in DMEM/F12 (Hyclone SH30023.01) containing 2 mM glutamine onice, The tissue was treated with 0.1% trypsin in Hanks' balanced saltsolution (HBSS, Gibco 14175-095) for 15 min at 37° C. and dissociatedmechanically by mild trituration with a fire-polished glass pipet ingrowth medium plus 2% heat-inactivated fetal bovine serum (FBS). Thegrowth medium consisted of equal volumes of Dulbecco's minimal essentialmedium DMEM (Gibco 12100-046) and HAM's F12 nutrient mixture (Gibco21700-075) supplemented with 33 mM glucose, 13 mM sodium bicarbonate, 5mM HEPES, 2 mM glutamine, 25 μg/ml insulin, 100 μg/ml transferrin, 60 μMputrescine, 20 nM progesterone, 30 nM sodium selenite, 5 U/ml penicillinG and 5 μg/ml streptomycin. 24-well tissue culture plate was coated byadding 15 μg/ml poly-1-ornithine hydrobromide solution at 0.5 ml/well,incubated at room temperature for 1 hr. The coating solution was removedand the well washed 3 times with distilled water and one wash with HBSS.The dissociated cells were plated onto polyornithine-coated wells at250,000-350,000 trypan blue-excluding viable cells per well in 500 μl ofabove medium containing 1% FBS. After 3 h when most of the cells hadadhered to the bottom of the well, the medium was replaced with 500 μlof fresh medium without FBS. A serial dilution of the sample to beassayed for trophic activity was added to each well in duplicate at thetime of medium change or within 24 h thereafter. Unless otherwisestated, cultures were incubated at 37° C. without any further mediumchange up to 7 days. Primary hindbrain cultures were prepared asdescribed for the midbrain cultures except that the rostral raphe region(instead of the rostral mesencephalon) was dissected and trypsinized for20 (instead of 15) min.

Dopamine (DA) Uptake Assay

[³H]DA uptake was measured in cultures at day 7, and all the solutionswere maintained at 37° C. The growth medium was removed, and thecultures were rinsed twice with 0.25 ml of uptake buffer, which consistsof HBSS (Gibco 11201-092) supplemented with 28 mM glucose, 15 mM HEPES,1 mM ascorbic acid (an antioxidant), and 0.5 nM pargyline (a monoamineoxidase inhibitor). The cultures were then incubated with 0.25 ml offresh uptake buffer containing 50 nM [³H]DA (NEN/DuPont) for 20 min at37° C. [³H]DA uptake was stopped by removing the incubation mixture, andcells were then washed twice with 0.5 ml of the uptake buffer at roomtemperature. To release [³H]DA from the cells, the cultures were lysedwith 0.2 ml of 0.1 N NaOH for 1 h at room temperature, the lysate wasthen added to 1 ml of Microscint-20 (Packard 6013621), and counted forradioactivity in a microplate scintillation counter (Packard TopCount,NXT). Background values were obtained by adding to the uptake buffer 0.5mM GBR-12909, a specific inhibitor of the high-affinity uptake pump ofthe dopaminergic neurons, and were usually <5% of the ³H uptake inuntreated control cultures. Forskolin (Fk, 25 μM) was employed as apositive control.

Immunocytochemistry

The primary antibodies were: anti-TH monoclonal (1:400,Boehringer-Mannheim); anti-GFAP polyclonal (1:200, DAKO); anti-NSEpolyclonal (1:2000, Polysciences Inc.); anti-tryptophan hydroxylasepolyclonal (TPH) (1:500, Eugene Tech) and anti-islet-1 monoclonal(1:100, Developmental Studies Hybridoma Bank). Cultures were fixed with4% paraformaldehyde in phosphate buffered saline (PBS), blocked with 3%bovine serum albumin plus 3% normal goat serum and permeabilized w/0.5%Triton in PBS. The primary antibodies were diluted in PBS containing 3%BSA and 0.4% Triton. Cells were visualized with the peroxidase-coupledavidin-biotin staining Vectastain ABC kit (Vector labs). Process-bearingTH-positive cells were counted at 100× magnification in consecutivefields covering the whole 16-mm-diameter culture well. GFAP-positivecells were counted in half the culture well. NSE-positive cells werecounted in 3 photographed fields.

Serotonin (5-HT) and 1-Aminobutyric Acid (GABA) Uptake Assays

Cells were incubated and treated as for measuring [³H]DA uptake with thefollowing exceptions. For measuring serotonin uptake, [³H]DA wasreplaced with 50 nM [³H]serotonin (Amersham), and background values wereobtained by adding 10 μM citalopram (a specific inhibitor of neuronalserotonin uptake). Fot measuring GABA uptake, the uptake buffer consistsof HBSS (Gibco 11201-092) containing 5.6 mM glucose and was supplementedwith 1.3 mM EDTA, 10 μM aminooxyacetic acid (a transaminase inhibitor toprevent GABA decomposition), 2 mM β-alanine (to inhibit glial uptake ofGABA) and 0.1 μM [¹⁴C]GABA (NEN/DuPont). Background values were obtainedby adding 1 mM diaminobutyric acid, a specific inhibitor of neuronalGABA uptake. FBS (2%) was added as a positive control.

Staining for Apoptotic DA Neurons

To measure the percent of DA neurons undergoing apoptosis afterdifferent treatments, DIV-13 cultures (which were medium changed twiceon DIV 5 and 9) were first stained with an anti-TH antibody and withFITC-conjugated goat anti-mouse secondary antibody (1:200, Jackson Lab)for 1 h at room temperature to label DA neurons. Cultures were thenincubated with the fluorescent DNA dye Hoechst 33258 (Sigma), at 4 μg/mlfor 5 min at room temperature, to visualize nuclear morphology. Cellswere viewed under an inverted fluorescence microscope (Olympus) equippedwith appropriate filters. Healthy and apoptotic TH⁺ cells TH⁺ cellnumbers were counted in the entire area of each well by an observer whois blinded to the treatment.

Statistical Analysis

Data are expressed as the means±SD. All experiments were repeated atleast two times. Data were analyzed by ANOVA, and the significance ofintergroup differences was determined by Student's t-test (two-tailed).For multiple comparisons, ANOVA followed by post hoc Dunnett's test wasperformed. Differences at p<0.05 were considered significant.

Results Identification of a Dopaminotrophic Small Molecule

Various natural product/small molecule libraries were tested in thedopamine uptake assay above to identify a neurotrophic activity formidbrain dopaminergic neurons. Dopamine uptake measures the number andactivity of specific dopamine reuptake transporters and reflects thefunctional differentiation of the dopaminergic neurons. Out of over10,000 compounds tested, we identified one, compound A, thatreproducibly stimulated DA uptake. The effect of compound A wasdose-dependent with a maximal effect at around 2.5 μM (FIG. 32A).Inclusion of the DA transporter inhibitor GBR-12909 abolished uptake,verifying that the observed uptake was specific to DA neurons, withouteffects on noradrenergic neurons, glial cells, or nonspecificabsorption. At concentrations above 10 μM, compound A was toxic andhence decreased DA uptake.

The percent stimulation by compound A remained about the same betweenDIV 5 and 7 (data not shown). In different experiments, the maximalstimulation by compound A on DIV 6 varied between 180-280%, comparableto that of GDNF in this assay (Lin et al. J Biol Chem 1994, 265,8942-8947). Compound A upregulated tyrosine hydroxylase (TH)immunoreactivity in mesencephalic cultures. More significantly, thenumber of TH positive neurons, a plausible index for dopaminergic neuronsurvival, also was increased by compound A dose-dependently in the sameconcentration range that it increased DA uptake (FIG. 32B). Adding 2.5-5μM of compound A once at the time of plating resulted in about twice asmany DA neurons at either day 7 or day 12. Consistent with thesefindings, TH enzyme and protein were elevated following compound Atreatment (data not shown).

Specificity

The effect of compound A in the midbrain culture appeared ratherselective for dopaminergic neurons relative to neurons generally,because visual inspection of compound A-treated cultures byphase-contrast microscopy did not reveal an obvious difference fromcontrol cultures with respect to neuronal density. Because TH-positiveneurons make up only ˜0.4% of the total population, an increase in theirnumber in the presence of compound A would not significantly affect thenumber of phase-contrast-bright cells. The visual observation wasconfirmed by total neuron immunocytochemical staining forneuron-specific enolase (NSE). NSE-positive cell numbers in the compoundA-treated wells were indistinguishable from the non-treated controlwells (FIG. 33A).

Compound A did not increase the density of astrocytes nor theirexpression of GFAP in the midbrain cultures. GFAP-positive cell numberswas not affected with the compound A treatment (FIG. 33B). Moreover,CUR-162590 had little, if any, effect on GABA-containing or serotonergicneurons, the two most abundant neuronal populations in the midbraincultures. Neither GABA (FIG. 33C) nor serotonin (FIG. 33D) uptake wassignificantly affected by compound A in the concentration range thatincreased DA uptake. These data suggest that compound A acts directlyand specifically on TH⁺ neurons.

Since the aforementioned bioactivities are reminiscent of those of GDNF(Lin et al. J Biol Chem 1994, 265, 8942-8947), and since GDNF alsoincreases the number of cranial motoneurons in these midbrain cultures(Zurn et al. Neuroreport 1994, 6, 113-118), we looked for this specificclass of neurons using antibody directed against islet-1 (Ericson et al.Science 1992, 256, 1555-1560). Compound A produced a small butsignificant, (115-150% of control) increase in the number of islet-1⁺motoneurons which constitute only ˜0.2% of the cells plated in theculture (FIG. 33E). Thus, like GDNF, compound A promotes the survival ofboth DA neurons and cranial motoneurons that, combined, account for <1%of the cells in these cultures.

The relative specificity of compound A in midbrain dopaminergic neuronswas further investigated in the embryonic hindbrain culture where raphenucleus containing the major serotonergic neurons reside. Compound A didnot promote the survival of neurons immunoreactive for the keyserotonin-synthesizing enzyme TPH (FIG. 33F), nor did it increaseserotonin uptake in the hindbrain culture at concentrations necessaryfor increasing dopamine uptake in the midbrain culture (FIG. 33G). At 10μM, compound A showed cytotoxicity on the hindbrain culture, asreflected in the inhibition of serotonin uptake, just as that observedin the midbrain culture in which compound A above 10 μM inhibited thedopamine uptake.

Effect of compound A on DA neuron apoptosis. To explore the mechanism bywhich Compound A promotes DA neuron survival, we compared the amount ofapoptosis of TH⁺ cells in cultures with and without compound Atreatment. We identified apoptotic TH⁺ cells by the appearance offragmented nuclei containing condensed chromatin as revealed by Hoechstdye (Clarkson et al. Neuroport 1995, 7, 145-149; Burke et al. JNeurochem 1998, 71, 517-525). We found that treatment with compound Areduced the rate of apoptosis by about 50% (FIG. 34A). Hence, the effectof compound A in enhancing DA neuron survival is mediated, at least inpart, by a reduction in apoptotic cell death.

Compound A and cell death induced by toxin treatment. Toxins and freeradicals have been implicated in the cell loss that occurs in PD(Gerlach and Riederer, J Neural Transm 1996, 103, 987-1041). Midbrain DAneurons are selectively vulnerable to MPTP (Mytilineou and Cohen,Science 1984, 225, 529-531), a neurotoxin that can cause the acute onsetof Parkinsonian symptoms in both humans and animals (Langston et al.Science 1983, 219, 979-980; Heikkila et al. Science 1984, 225,1451-1453). Having shown that compound A increases the survival ofcultured DA neurons, we investigated whether compound A could alsoreduce the susceptibility of these neurons to MPP⁺, the activemetabolite of MPTP. Exposure of control cultures to 2 μM MPP⁺ for 36 hdestroyed 48% of the TH⁺ neurons. However, the neuronal loss was reducedto 20% in compound A-treated cultures (FIG. 34B). compound A mostlyprevented this loss (Fig. These data indicate that compound A protectsDA neurons them from neurotoxin-induced degeneration.

Structurally Related Compounds

Several compounds (Compounds B-B″) which are structurally similar tothat of compound A were found to stimulate dopamine uptake in themidbrain culture. The stimulation was dose dependent between 2.5 and 20μM. The activity profiles of three active analogues are shown in FIG.36: compound X′ and compound Y′ showed no cytotoxicity even at 20 μM,compound Z′ showed a higher stimulation maximum, and all three had lowerhalf-maximal effective concentrations (EC50) of about 0.2 μM. Theseobservations suggest that compound A may represent a prototype of aclass of molecules that can be optimized via medicinal chemistry to geta pharmacologically desirable drug to treat certain neurodegenerativediseases such as PD.

Additive/Synergistic Effect of Compound A and GDNF

Since the GDNF effect on DA uptake plateaus around 1 ng/ml (Lin et al.Science 1993, 260, 1130-1132; Lin et al. J. Neurochem 1994, 63,758-768), we added 2.5 μM compound A to increasing concentrations ofGDNF. The addition of compound A to a maximally effective concentrationof GDNF increased DA uptake above that reached by either GDNF orcompound A alone (FIG. 37). The combined effect is at least as potent asthat of forskolin, a cAMP stimulator used as positive control in theassay. Potentially, conjoint administration of compound A (or othersubject compounds) with GDNF (or related factors) may provide aneffective therapy for treating Parkinson's disease, allowing eachcompound to be administered in a lower dose in combination than would berequired for either alone, thereby reducing any undesirable side effectsthat may be associated with dosing either compound at the therapeuticdose required for administration alone.

All of the above-cited references and publications are herebyincorporated by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A kit for treating neurodegenerative diseases such as Parkinson'sdisease and amyotrophic lateral sclerosis (ALS), comprising a compoundof Formula I:

wherein, as valence and stability permit, M_(i) is absent or represents—CH═CH—; T and X, independently, are absent; V and Z, independently,represent —N(R)—, —O—, —S—, or —Se—; R represents H or lower alkyl; Uand Y, independently, represent —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, or amethylene group optionally substituted with 1-2 C1-C2 lower alkylgroups; R₁ represents a substituted or unsubstituted alkyl, heteroalkyl,carbocyclic aliphatic, heterocyclic aliphatic, aryl, or heteroarylgroup; Ar represents a substituted or unsubstituted aryl or heteroarylgroup; Ht is selected from:


2. The kit according to claim 1 wherein Ht is


3. The kit according to claim 1 wherein Ht is selected from:


4. The kit according to claim 1 wherein Ht is selected from:


5. The kit according to claim 1, wherein the compound of Formula I isselected from the group consisting of:


6. The kit according to claim 1, wherein the compound of Formula I isselected from the group consisting of:


7. The kit according to claim 1, wherein the compound of Formula I isselected from the group consisting of:


8. A kit for treating neurodegenerative diseases such as Parkinson'sdisease and amyotrophic lateral sclerosis (ALS), comprising a compoundof Formula I:

wherein, as valence and stability permit, M_(i) represents —CH═CH—; Tand X, independently, are absent; V and Z, independently, represent—N(R)—, —O—, —S—, or —Se—; R represents H or lower alkyl; U and Y,independently, represent —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, or amethylene group optionally substituted with 1-2 C1-C2 lower alkylgroups; R₁ represents a substituted or unsubstituted alkyl, heteroalkyl,carbocyclic aliphatic, heterocyclic aliphatic, aryl, or heteroarylgroup; Ar represents a substituted or unsubstituted aryl or heteroarylgroup; Ht is selected from:


9. The kit according to claim 8 wherein Ht is


10. The kit according to claim 8 wherein Ht is selected from:


11. The kit according to claim 8 wherein Ht is selected from:


12. The kit according to claim 8 wherein Ht is selected from:


13. The kit according to claim 8, wherein the compound of Formula I isselected from the group consisting of:


14. The kit according to claim 8, wherein the compound of Formula I isselected from the group consisting of:


15. The kit according to claim 8, wherein the compound of Formula I isselected from the group consisting of:


16. The kit according to claim 8, wherein the compound of Formula I isselected from the group consisting of:


17. A kit for treating neurodegenerative diseases such as Parkinson'sdisease and amyotrophic lateral sclerosis (ALS), comprising a compoundof Formula IV:

wherein, as valence and stability permit, M represents, independentlyfor each occurrence, a heteroatom or a substituted or unsubstitutedmethylene group, such as —CH₂—, —CHF—, —CHOH—, —CH(Me)—, —C(═O)—, etc.,or two M taken together represent substituted or unsubstituted ethene orethyne; Q represents, independently for each occurrence, O or S; R,independently for each occurrence, represents H or lower alkyl; T and V,independently, are absent or represent —N(R)—, —O—, —S—, or —Se—; U andY, independently, represent —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, or amethylene group optionally substituted with 1-2 C1-C2 lower alkylgroups; X and Z, independently, are absent or represent —N(R)—, —O—,—S—, or —Se—; Cy represents a substituted or unsubstituted carbocyclicaliphatic, heterocyclic aliphatic, aryl, or heteroaryl group; Arrepresents a substituted or unsubstituted aryl or heteroaryl group; Htrepresents a substituted or unsubstituted heterocyclic aliphatic orheteroaryl group; and i represents an integer from 0 to
 4. 18. The kitaccording to claim 17, wherein the compound of Formula IV is selectedfrom the group consisting of:


19. The kit according to claim 1, wherein the disease is psychosis suchas schizophrenia.
 20. The kit according to claim 1, wherein the diseaseis peripheral neuropathy, acquired neuropathies such as diabeticneuropathies, or immune-mediated neuropathies.